JP6925608B2 - Cross-section processing observation method, cross-section processing observation device - Google Patents

Description

In the present invention, a cross-sectional processing observation is performed to obtain a cross-sectional image by irradiating a specific site including a specific observation object of a sample formed by a focused ion beam with an electron beam to obtain a three-dimensional stereoscopic image of the specific observation object. It relates to a method and a cross-section processing observation device.

For example, as one of the methods for analyzing the internal structure of a sample such as a semiconductor device or performing three-dimensional observation, a focused ion beam (FIB) lens barrel and an electron beam (EB) are used. Using a composite charged particle beam device equipped with a lens barrel, cross-section forming by FIB and scanning electron microscope (Scanning Electron Mi) by EB scanning the cross section
A cross-sectional processing observation method is known in which a plurality of cross-sectional images of a target sample are obtained by repeating croscope (SEM) observation, and then the plurality of cross-sectional images are superposed to construct a three-dimensional stereoscopic image of the sample (for example). , Patent Document 1).

In this cross-section processing observation method, cross-section formation processing by FIB is referred to as Cut, cross-section observation by EB is referred to as See, and a series of methods for constructing a three-dimensional image by repeating Cut and See is known as Cut & See. In this method, the three-dimensional shape of the target sample can be seen from various directions from the constructed three-dimensional image. Furthermore, it has an advantage that other methods do not have, that is, it is possible to reproduce an arbitrary cross-sectional image of the target sample.

As a specific example, the sample is irradiated with FIB and etched to expose the cross section of the sample. Subsequently, the exposed cross section is observed by SEM to obtain a cross-sectional image. continue,
After etching again to expose the next cross section, a second cross-sectional image is obtained by SEM observation. In this way, etching processing and SEM observation are repeated along an arbitrary direction of the sample to obtain a plurality of cross-sectional images. Finally, there is known a method of constructing a three-dimensional stereoscopic image in which the inside of the sample is transmitted by superimposing a plurality of acquired cross-sectional images.

Japanese Unexamined Patent Publication No. 2008-2700073

However, for example, when the sample to be observed is a biological sample such as a cell, it is embedded with an embedding agent such as paraffin and fixed to the embedding block. When such a biological sample is used as a processing observation target and a high-resolution three-dimensional stereoscopic image is selectively constructed only on a specific observation target such as minute cells dispersed in the target sample, the embedding block is formed by FIB or EB. When observing, only the surface layer of the embedding block is observed, and the existence position of a specific observation object such as cells dispersed in the target sample (embedding block) cannot be specified. Therefore, when the above-mentioned Cut & See method is applied to a sample in which specific observation objects are scattered, a processing region by FIB is set for the entire sample, and Cut and See are repeatedly performed for the entire sample to perform the specific observation object. It is necessary to make the sample appear in the processed cross section, and there is a problem that it takes an enormous amount of time for observation.

Further, when Cut and See are repeatedly performed on the entire sample, the number of cross-sectional images generated becomes enormous. Therefore, in order to generate a three-dimensional stereoscopic image of a specific observation object, only a cross-sectional image of a part including the specific observation object is extracted from such a large number of cross-sectional images to construct a three-dimensional stereoscopic image. There is also the problem that inefficient and time-consuming procedures are required. In particular, in the case of biological samples, the size of the specific observation object is very small in most cases, and in order to obtain a highly accurate three-dimensional image, the processing interval by the FIB is made as narrow as possible to widen the field of view. It is necessary to repeat the SEM observation of. Therefore, a large amount of cross-sectional images are acquired for the entire sample, but a cross-sectional image other than a specific part including a specific observation object is unnecessary, and image processing is performed to process or temporarily store such an unnecessary cross-sectional image. There is also the problem that the device is overloaded.

Furthermore, if the specific observation object is not included in the sample, all the observations by Cut and See of the entire sample, which took a long time, are wasted, which is an obstacle in efficient observation. There was also.

The present invention has been made in view of the above-mentioned circumstances, and enables quick and efficient observation of only a specific part including a specific observation object contained in a sample, so that the specific observation object 3 It is an object of the present invention to provide a cross-section processing observation method and a cross-section processing observation device capable of easily forming a three-dimensional stereoscopic image.

In order to solve the above problems, some aspects of the present invention provide the following cross-section processing observation method and cross-section processing observation device.
In other words, cross-section processing and observation method of the present invention is to observe the entire sample to be observed with an optical microscope, to obtain an approximate three-dimensional position coordinate information within said sample of a particular observation object contained in the sample position Based on the information acquisition process and the three-dimensional position coordinate information, cross-sectional processing that irradiates a focused ion beam toward a specific part of the sample in which the specific observation object exists to expose the cross section of the specific part. The step, the cross-sectional image acquisition step of irradiating the cross section with an electron beam to acquire an image of a region of a predetermined size including the specific observation object, and the cross-sectional processing step and the cross-sectional image acquisition step are performed at predetermined intervals. in repeated a plurality of times along a predetermined direction, and having a three-dimensional image generating step of constructing a three-dimensional image including the specific observation object from a plurality of pre-outs image obtained, the.

In the cross-sectional processing observation method of the present invention, the sample to be observed is observed in advance with an optical microscope tube, and the position coordinate information in the XYZ direction in which the specific observation object is present is acquired and stored. The position coordinates in the Z (depth) direction by the optical microscope tube are obtained by observing while changing the focal position by fixing the XY position coordinates using a confocal stereomicroscope as the optical microscope tube.

Based on the three-dimensional coordinate information of the specific observation object thus obtained, the sample to be observed is subjected to cross-section exposure processing by FIB. At this time, the region where the specific observation object does not exist is processed in a short time by widening the processing interval by the FIB, and the cross-sectional image is not particularly acquired. Then, when the specific portion including the specific observation object is reached by the position coordinate information, the sample is processed at a predetermined narrow interval, and the SEM cross-sectional image of the region including the specific observation object in the processed cross section is acquired.

Then, the processing position of the FIB and the SEM image are saved as a set. When the acquisition of the image data of the specific part including the specific observation object is completed, the acquisition of the cross-sectional image and the FIB processing at a narrow interval are completed, and the FIB processing is performed at a wide interval in a short time until the next specific part is approached. When the cross-sectional images of all the specific parts can be obtained in this way, the processing is completed. Alternatively, the cross-sectional image is not acquired in the region where the processing interval is constant and there is no object, but the cross-sectional image is acquired in the region where the object is present. Thereby, desired information can be efficiently acquired.

As described above, according to the cross-sectional processing observation method of the present invention, by grasping the coordinate position where the specific observation object exists by optical observation with the optical microscope tube, the specific observation can be performed quickly and efficiently. By observing a specific part including an object, it is possible to easily obtain a three-dimensional stereoscopic image of the specific observation object.

The present invention is characterized in that a plurality of types of the specific observation object are set, and the cross-section processing step is performed for each specific observation object.
Further, the present invention is characterized in that a confocal stereomicroscope is used as the optical microscope.

Further, in the present invention, in the cross-sectional image acquisition step, a cross-sectional composition image of the specific portion including the specific observation object is further acquired by energy-dispersed X-ray detection of the cross section, and is acquired in the stereoscopic image generation step. It is characterized in that a three-dimensional three-dimensional composition image including the specific observation object is constructed from the plurality of cross-sectional composition images.

The cross-sectional processing observation apparatus of the present invention includes a sample table on which a sample containing a specific observation object is placed, a focused ion beam lens barrel that irradiates the sample with a focused ion beam, and an electron beam that irradiates the sample with an electron beam. By the lens barrel, an optical microscope barrel for optically observing the sample, a secondary electron detector for detecting secondary electrons generated from the sample, a reflected electron detector for detecting reflected electrons, and the optical microscope barrel. The position of the specific observation object in the sample is specified, and the focused ion beam is irradiated from the focused ion beam barrel toward the specific part of the sample in which the specific observation object is present to obtain a cross section. It is exposed, and the cross section is irradiated with an electron beam from the electron beam lens barrel to acquire an image of a region of a predetermined size including the specific observation object, and the image of the specific portion including the specific observation object is obtained. It is characterized by having a control unit for constructing a three-dimensional stereoscopic image.

According to the cross-sectional processing observation device of the present invention, the approximate position coordinates of the specific part including the specific observation object in the sample can be grasped in advance by the optical microscope tube, so that the specific part is processed when the sample is processed by the focused ion beam lens barrel. The region other than the specific region is rough, and the specific region can be finely processed with a focused ion beam tube. As a result, a high-resolution cross-sectional image of a specific part including a specific observation object can be quickly obtained in a short time.

Further, according to the cross-section processing observation apparatus of the present invention, among the cross-sections of the sample formed by the focused ion beam barrel, only a specific part including the specific observation object is irradiated with an electron beam from the electron beam barrel to form a cross-section. In order to acquire image data, the data capacity of the cross-sectional image to be stored is significantly reduced compared to the conventional observation device that obtains a cross-sectional image over the entire sample, and a cross-section processing observation device can be configured at low cost. Can be done.
Furthermore, when acquiring a cross-sectional image with an electron beam of a specific part including a specific observation object, it is possible to obtain a cross-sectional image over the entire sample by imaging with high resolution, as compared with a conventional observation device. It becomes possible to generate a three-dimensional stereoscopic image with high resolution.

Further, the present invention is characterized in that an EDS detector for detecting characteristic X-rays generated from the sample is provided, and the control unit constructs a three-dimensional three-dimensional composition image of the specific portion.

According to the present invention, it is possible to process a specific part including a specific observation object with high accuracy in a short time, thereby easily and quickly producing a high-resolution three-dimensional stereoscopic image of the specific observation object. It becomes possible to generate.

It is a schematic block diagram which shows the cross-section processing observation apparatus of 1st Embodiment. It is a flowchart which showed one Embodiment of the cross-section processing observation method step by step. It is a schematic diagram when observing a sample with an optical microscope tube. It is a schematic diagram which shows the specific part which becomes the acquisition area of the SEM image. It is a flowchart which shows the modification of the processing observation method. It is a schematic block diagram which shows the cross-section processing observation apparatus of 2nd Embodiment. It is a schematic block diagram which shows the cross-section processing observation apparatus of 3rd Embodiment. It is a projection drawing which concerns on the cross-section processing observation method of 2nd Embodiment. It is a projection drawing which concerns on the cross-section processing observation method of 3rd Embodiment. It is a schematic block diagram which shows the cross-section processing observation apparatus of 4th Embodiment. It is explanatory drawing which shows the cross-section processing observation method of 4th Embodiment. It is explanatory drawing which shows the cross-section processing observation method of 4th Embodiment. It is explanatory drawing which shows the cross-section processing observation method of 4th Embodiment. It is explanatory drawing which shows the cross-section processing observation method of 4th Embodiment.

Hereinafter, the cross-section processing observation method and the cross-section processing observation device of the present invention will be described with reference to the drawings. It should be noted that each of the embodiments shown below is specifically described in order to better understand the gist of the invention, and is not limited to the present invention unless otherwise specified. In addition, the drawings used in the following description may be shown by enlarging the main parts for convenience in order to make the features of the present invention easy to understand, and the dimensional ratios of the respective components are the same as the actual ones. Is not always the case.

(Cross-section processing observation device: first embodiment)
FIG. 1 is a schematic configuration diagram showing a cross-section processing observation device of the first embodiment.
The cross-section processing observation device 10 of the present embodiment includes a focused ion beam (FIB) lens barrel 11, an electron beam (EB) lens barrel 12, an optical microscope (ОМ) tube 13, and a stage (sample table) 15. It has at least a sample chamber 14. The focused ion beam barrel 11, the electron beam barrel 12, and the optical microscope barrel (optical microscope) 13 are fixed to the sample chamber 14, respectively.

The focused ion beam barrel 11, the electron beam barrel 12, and the optical microscope barrel 13 are directed toward the sample S placed on the stage 15, and the focused ion beam (FIB) 21, the electron beam (EB) 22, and the visible beam (EB) 22 are visible. The light (VL) 23 is arranged so that it can be irradiated.

Focused ion beam barrel 11 and electron beam mirror so that the focused ion beam 21 emitted from the focused ion beam barrel 11 and the electron beam 22 emitted from the electron beam barrel 12 are orthogonal to each other on the sample S. It is more preferable to arrange the cylinder 12 because the electron beam can be irradiated perpendicularly to the processed cross section and a high-resolution cross-sectional image can be obtained.

Here, as an example of the sample S, a biological sample embedded in a resin or the like can be mentioned. The observer pays particular attention to observing, for example, cells contained (encapsulated) in such a biological sample as a specific observation object (part of interest). When the sample is a transparent biological sample, unlike general materials such as metals, the inside of the biological sample can be observed with an optical microscope tube (optical microscope) 13, so that the position of the attention portion can be confirmed. ..

The optical microscope cylinder (optical microscope) 13 is arranged so that it can be observed in substantially the same direction as the irradiation direction of the electron beam 22. A part or all of the optical microscope cylinder 13 may be housed in the sample chamber 14, or may be arranged in another sample chamber arranged separately from the sample chamber 14. In any case, it is preferable to arrange the electron beam so that it can be observed in substantially the same direction as the irradiation direction of the electron beam.

For the optical microscope cylinder (optical microscope) 13, for example, it is particularly preferable to use a confocal stereomicroscope provided with a laser light source as the light source. The confocal stereomicroscope can image the sample S in the height (depth) direction (direction along the optical axis of the optical microscope) with high resolution. A confocal stereomicroscope is mostly used to image a sample having minute irregularities on its surface, but in the present embodiment, the internal structure of the sample S is oriented in the depth direction. It is used to acquire a high-resolution image along the line.

For example, in a transparent sample S such as a biological sample, the laser beam is transmitted, and if there is a foreign substance or tissue in the sample S that does not transmit the laser beam, they can be imaged. As is well known, the XY plane image information of the sample S is performed by laser scanning by a scanning mechanism that two-dimensionally scans the sample S along the respective directions of the X-axis and the Y-axis, and the height (depth) of the sample is obtained. As for the information in the direction (Z axis), a three-dimensional image is acquired by capturing the Z position (height information of the sample S) when the brightness is maximum.

However, in the image obtained by the optical microscope tube (optical microscope) 13, the structure behind the imaged specific observation object cannot be known because the specific observation object is opaque. In this way, only the shape of the uppermost surface on which the laser beam is reflected can be known, but since the in-plane position information (XY coordinates) and the depth position information (Z coordinates) where the specific observation object exists can be obtained, Cut & Seee By the operation, it is possible to establish a guideline as to where the specific observation object exists in the sample S.

On the other hand, in the case of observation using the focused ion beam 21 or the electron beam 22, even if the sample S is transparent, only the morphological information of the outermost surface of the sample S can be obtained. Therefore, even if it is known in advance that the specific observation object exists inside the sample S, its existence position (coordinates) cannot be clarified. Further, it may be difficult to grasp the attention portion in the SEM image due to the difference in contrast between the optical microscope image and the SEM image.

Therefore, by observing with an optical microscope using an optical microscope tube (optical microscope) 13, the coordinates of a specific part, which is a small region in the sample S including the specific observation object, are set prior to the Cut operation (cross-section processing step) of the sample S. Can be obtained. Then, the Cut operation (cross-section processing step) is carried out based on the coordinates of the specific part including the specific observation object, but in order to ensure the acquisition region of the SEM image by the electron beam 21 for the exposed cross-section, the cross-section is optical. If there is a characteristic form that can be commonly recognized by the microscope tube (optical microscope) 13 and the electron beam 21, the position (coordinates) of the specific observation object can be specified from the relative positional relationship using this as a reference point.

The stage 15 is controlled by the stage control unit 19, and the sample S can be placed on the stage 15 and can be moved and tilted in each direction of the XYZ, whereby the sample S can be adjusted in any direction.

The cross-section processing observation device 10 further includes a focused ion beam (FIB) control unit 16, an electron beam (EB) control unit 17, and an optical microscope (ОМ) control unit 18, respectively. The focused ion beam control unit 16 controls the focused ion beam lens barrel 11 to irradiate the focused ion beam 21 at an arbitrary timing. The electron beam control unit 17 controls the electron beam lens barrel 12 to irradiate the electron beam 22 at an arbitrary timing.

The optical microscope control unit 18 controls the optical microscope cylinder 13 to move the position of the optical focal point, and acquires and stores the observation image of the sample S each time the optical focus is moved. The focal position is repeatedly finely moved and stopped in one direction of gradually approaching or moving away from the optical microscope cylinder 13 along the optical axis of the optical microscope cylinder 13, and an image of the sample S is acquired at the time of stopping. Alternatively, the focal position of the optical microscope cylinder 13 may be fixed, and the stage 15 may be finely moved in one vertical direction by the stage control unit 19 to acquire an optical microscope image.

The cross-section processing observation device 10 further includes a secondary electron detector 20. The secondary electron detector 20 irradiates the sample S with the focused ion beam 21 or the electron beam 22, and detects the secondary electrons generated from the sample S.

It is also preferable to provide a backscattered electron detector (not shown) in place of the secondary electron detector 20 or in addition to the secondary electron detector 20. The backscattered electron detector detects the backscattered electrons reflected by the electron beam on the sample S. A cross-sectional image of the sample S can be obtained by such reflected electrons.

The cross-section processing observation device 10 includes an image forming unit 24 for forming an observation image of the cross section of the sample S, and a display unit 25 for displaying the observation image. The image forming unit 24 forms an SEM image from the signal for scanning the electron beam 22 and the signal of the secondary electrons detected by the secondary electron detector 20. The display unit 25 displays the SEM image obtained by the image forming unit 24. The display unit 25 may be composed of, for example, a display device.

The cross-section processing observation device 10 further includes a control unit 26 and an input unit 27. The operator inputs various control conditions of the cross-section processing observation device 10 via the input unit 27. The input unit 27 transmits the input information to the control unit 26. The control unit 26 outputs a control signal to the focused ion beam control unit 16, the electron beam control unit 17, the optical microscope control unit 18, the stage control unit 19, the image forming unit 24, and the like, and operates the entire cross-section processing observation device 10. Control.

(Cross-section processing observation device: second embodiment)
In the first embodiment described above, a configuration in which a part or all of the optical microscope tube (optical microscope) 13 is inside the sample chamber 14 has been described, but the optical microscope tube (optical microscope) 13 is installed outside the sample chamber 14. You may.
FIG. 6 is a schematic configuration diagram showing a cross-section processing observation device according to a second embodiment of the present invention. The same configuration as that of the first embodiment is assigned the same number, and duplicate description will be omitted. In the cross-sectional processing observation device 30, a second sample chamber 34 is formed outside the sample chamber 14, and an optical microscope cylinder (optical microscope) 33 is arranged in the second sample chamber 34.

The inside of the second sample chamber 34 in which a part or all of the optical microscope cylinder (optical microscope) 33 is installed is evacuated or atmospheric pressure. The inside of the sample chamber 14 to which the focused ion beam or the electron beam is irradiated must be a vacuum, but since the installation environment of the optical microscope cylinder (optical microscope) 33 may be under atmospheric pressure, the second sample chamber 34 is used. By creating an atmospheric pressure environment, the volume of the sample chamber 14 that becomes a vacuum can be reduced, and the efficiency of vacuum exhaust can be improved.

The movement of the sample S between the sample chamber 14 and the second sample chamber 34 is performed by the transport rod 36 together with the sample holder 35 on which the sample S is placed. When moving, the transport rod 36 is fixed to the sample holder 35, the airtight door (valve) 37 is opened, and then the sample holder 35 removed from the stage 38 of the second sample chamber 34 is pushed into the sample chamber 14 by the transport rod 36. , The sample holder 35 is fixed on the stage 15 in the sample chamber 14.

The stage 15 of the sample chamber 14 and the stage 38 of the second sample chamber 34 provided with the optical microscope cylinder (optical microscope) 33 are controlled by the stage control units 39a and 39b, respectively. The coordinate information in the stages 15 and 38 is stored in the control unit 26, and the stage 15 in the sample chamber 14 can be linked based on the coordinate information obtained in the stage 38 in the second sample chamber 34. Although the focused ion beam control unit and the electron beam control unit are omitted in FIG. 6, the configuration is the same as that in FIG.

(Cross-section processing observation device: third embodiment)
In the second embodiment described above, the configuration in which the second sample chamber 34 in which the optical microscope cylinder (optical microscope) 13 is provided separately from the sample chamber 14 is arranged has been described, but the sample chamber in which the optical microscope cylinder (optical microscope) 13 is installed has been described. Does not have to be formed.
FIG. 7 is a schematic configuration diagram showing a cross-section processing observation device according to a third embodiment of the present invention. The same number will be assigned to the same configuration as in the second embodiment, and duplicate description will be omitted. In the cross-section processing observation device 40, an optical microscope cylinder (optical microscope) 43 is independently arranged outside the sample chamber 14. Then, the coordinates of the specific observation object (attention portion) in the sample S obtained by the optical microscope cylinder (optical microscope) 43 can be read by the focused ion beam control unit and the electron beam control unit (see FIG. 1). It has a partially shared control unit 46. By providing the optical microscope cylinder (optical microscope) 43 independently outside the sample chamber 14, the degree of freedom in the installation surface of the equipment can be increased, for example, by installing each of them in a separate chamber for work.

(Cross-section processing observation device: Fourth embodiment)
FIG. 10 is a schematic configuration diagram showing a cross-sectional processing observation device according to a fourth embodiment of the present invention. The same configuration as that of the first embodiment is assigned the same number, and duplicate description will be omitted. The cross-section processing observation device 50 includes an EDS detector 51 that detects X-rays generated from the sample S when the sample S is irradiated with an electron beam (EB), and an EDS control unit 52 that controls the EDS detector 51. It has. The X-rays generated from the sample S include characteristic X-rays peculiar to each substance constituting the sample S, and the substance (composition) constituting the sample S can be specified by such characteristic X-rays.

The image forming unit 24 forms a composition image of a specific cross section of the sample S from the scanning signal of the electron beam 22 and the signal of the characteristic X-ray detected by the EDS detector 51. The control unit 26 constructs a three-dimensional stereoscopic composition image based on a plurality of composition images. The composition image (EDS image) identifies the substance of the sample S at each electron beam irradiation point from the detected characteristic X-ray energy, and shows the distribution of the substance in the irradiation region of the electron beam 22.

By using the cross-section processing observation device 50 of the fourth embodiment, it is possible to construct a three-dimensional composition map of a specific part in the sample S. A method of constructing a three-dimensional composition map using the cross-section processing observation device 50 of the fourth embodiment will be described in the fourth embodiment of the cross-section processing observation method described later.

The cross-sectional processing observation device 50 of the fourth embodiment includes the optical microscope cylinder 13 and the secondary electron detector 20 together with the EDS detector 51, but the optical microscope cylinder 13 and the secondary electron detector 20 are omitted. You can also do it.

(Cross-section processing observation method: first embodiment)
Next, the cross-section processing observation method of the first embodiment of the present invention using the cross-section processing observation device described above will be described with reference to FIGS. 1 and 2.
FIG. 2 is a flowchart showing one embodiment of the cross-section processing observation method step by step.
First, the target sample S is observed with an optical microscope tube (optical microscope) 13, and the position (XYZ coordinates) information of the specific observation target (attention portion) contained in the sample S is acquired (position information acquisition step). S10). Sample S is, for example, a biological sample. The target sample S is embedded and fixed in advance with a resin or the like, and has been subjected to treatments such as drying and dehydration. A known technique may be used for the biological sample preparation steps such as the embedding work and the dehydration work.

When the sample S treated in this way is observed by SEM, an image of the surface of the sample S can be obtained, but an image of the inside of the sample S cannot be obtained. However, when observed with an optical microscope tube (optical microscope) 13, light is transmitted in the transparent sample S such as a living body, and a specific observation object (part of interest) such as a foreign substance or a tissue that does not transmit light is present in the sample S. , Can be visualized by the difference in contrast.

FIG. 3A is an optical microscope image (schematic diagram) when the sample S is observed by the optical microscope cylinder 13. It is a projection drawing of the image observed by changing the focal position from the surface of the sample S to a predetermined depth, and the specific observation objects (parts of interest) C1 and C2 appearing in the visual field are displayed on one screen. As the XY coordinates of the optical microscope image shown in the lower left of FIG. 3A, the approximate center coordinates of the specific observation object C1 are (X1, Y1), and the approximate center coordinates of the specific observation object C2 are (X2, Y2). Is.

FIG. 3B is an optical microscope image (schematic diagram) showing the depth direction of the same field of view of the same sample S as in FIG. 3A. The depth direction of the sample is the Z coordinate, and the area from the surface Z0 of the sample S to the observation bottom surface Zn is displayed. The focal point of the optical microscope cylinder 13 is sequentially adjusted from the surface toward the inside of the sample S (depth direction Z), and the optical microscope image at each focal position is stored in the control unit 26 together with the Z coordinate at that time.

Then, the acquisition of the optical microscope image (planar image) by the optical microscope cylinder 13 is repeated from the surface of the sample S to the predetermined observation bottom surface Zn. By synthesizing (image processing) a plurality of optical microscope images (planar images) thus obtained, a three-dimensional image of the sample S is reproduced, and the depths of the specific observation objects (parts of interest) C1 and C2 in the sample S are reproduced. The existing position (coordinates) of the direction Z can be grasped. In this way, by observing the sample S while fixing the XY coordinates and changing the optical focal position using the optical microscope cylinder (optical microscope) 13, the specific observation object (the portion of interest) in the sample S can be observed. Approximate three-dimensional coordinates can be clarified.

Next, based on the XY coordinate information of the specific observation object (part of interest) obtained by the optical microscope tube (optical microscope) 13, the focused ion beam 21 is irradiated from the focused ion beam lens barrel 11 to become a target. Cross-section forming processing of sample S is performed (cross-section processing step S11).
FIG. 4A is a schematic diagram showing specific parts (image acquisition areas) serving as SEM image acquisition areas for the specific observation object C1 and the specific observation object C2 shown in FIG. 3A. In the case of FIG. 4A, the image acquisition region (specific part) of the specific observation object C1 is the outermost part of the specific observation object, centered on (X1, Y1), which is approximately the center coordinates of the specific observation object C1. The length obtained by adding a predetermined distance from the end portion is defined as a rectangular region having one side ΔX1 and ΔY1. Similarly, the image acquisition region of the specific observation object C2 is a rectangle whose sides are ΔX2 and ΔY2, with the coordinates (X2, Y2) as the center and the length obtained by adding a predetermined distance from the outermost end of the specific observation object. It is an area.

This predetermined length is a margin area when the area including the specific observation object is displayed in three dimensions, and if this margin area is excessively large, the three-dimensional image of the specific observation object becomes small and there is no margin. If it exceeds the limit, it may be difficult to see depending on the display direction of the cross section. Therefore, the operator may appropriately determine this margin area in advance according to the appearance of the specific observation object. For example, the image acquisition range may be an increase of about 10% in each length of the projection drawing of the specific observation object in the XY directions.

FIG. 4B is a projection drawing of the sample S in the depth direction Z, and from this image, a processing region for exposing the cross section of the sample S at narrow intervals is determined by the focused ion beam 21. Here, in FIG. 4B, the appearance position where the specific observation object C1 appears in the observation field (upper part of the specific observation object) is Z11, and the disappearance position where the specific observation object C1 disappears from the observation field (specific observation object). (Lower part) is Z12, and similarly, the appearance position Z21 of the specific observation object C2 and the disappearance position of the specific observation object C2 are Z22.

The range in which the sample S is thinly processed by the focused ion beam 21 at close intervals is determined, for example, as the length obtained by adding a predetermined length to the appearance coordinates and the disappearance position of the specific observation object. The predetermined length is a margin area when the portion including the specific observation object is three-dimensionally displayed, and the Z direction is also, for example, 10% of the Z direction length (Z12-Z11) of the specific observation object C1. Is a margin area, and the Z coordinate Z1A to Z1B, which is the upper part of the Z coordinate Z11 and the lower part of the Z coordinate Z12, is the processing area. Similarly, for the specific observation object C2, the Z coordinates Z2A to Z2B are set as a cross-section processing range in which the sample S is processed at close equal intervals by the focused ion beam 21.

The narrower the processing interval of the sample S by the focused ion beam 21, the higher the resolution in the Z direction of the three-dimensional image constructed by the specific observation object. On the other hand, when the specific observation object is extremely long in the Z direction, the number of SEM images acquired increases, and the accumulated image data increases. Therefore, it is necessary to grasp the size of the specific observation object in advance by observing with the optical microscope tube (optical microscope) 13 and to obtain the accumulated image data capacity and the three-dimensional image of the specific observation object to be constructed. It is preferable to determine the processing interval by the focused ion beam 21 after examining the resolution in advance.

Cross-section processing by the focused ion beam 21 may be performed at a coarse interval between Z0 to Z1A, Z1B to Z2A, and Z2B to Zn on the Z coordinate outside the processing range at such a narrow interval. Further, since it is known that there is no specific observation object in this region, it is not necessary to dare to acquire the SEM image. As a result, it is possible to shorten the cross-sectional processing time of the sample S and reduce the storage capacity of the SEM image. Alternatively, it is possible to shorten the time and reduce the storage capacity by processing the cross section at regular intervals, acquiring an image when a specific observation object exists, and not acquiring an image when the object does not exist. can.

Next, among the cross-sections of the processed sample S, an SEM image of a specific part including a specific observation object is acquired and stored together with the cross-sectional position information (cross-sectional image acquisition step S12). Based on the coordinates of the existence position of the specific observation object obtained by the observation with the optical microscope cylinder (optical microscope) 13, an SEM image of a specific part which is a small area including the coordinates of the specific observation object is acquired. Then, the processing position by the focused ion beam 21, the SEM image, and the XY coordinates in the cross section (for example, the coordinates of the center of the SEM image) are stored as a set. For example, when a plurality of specific observation objects are present in one cross section, an SEM image of a specific part including each specific observation object is acquired.

Next, it is determined whether or not the image acquisition of the specific observation object is completed (S13). It is determined whether or not the processing by the focused ion beam 21 has reached the predetermined Z coordinate (for example, Z1B) at the end of processing. Then, when it is determined that the predetermined arrival position has been reached, the acquisition of the image data of the specific portion including the specific observation object is completed.

Then, the image acquisition and the processing by the focused ion beam 21 at a narrow interval are completed, and the cross-sectional processing by the focused ion beam 21 is performed at a wide interval until the area where the next specific observation object exists is approached. If there is no next specific observation object, the processing is completed.

In this way, by observing with the optical microscope tube (optical microscope) 13, the existing position (coordinates) of the specific observation object in the sample can be grasped in advance, so that the processing until the specific part including the specific observation object is reached. Can be performed quickly with a wide processing width. Further, since the image is not acquired in the portion where the specific observation object does not exist, the observation time can be minimized and the storage area of the image can be reduced. In addition, cross-sectional information can be obtained in more detail. Further, even if the processing width is constant, the presence or absence of image acquisition can be set, so that the time and storage area can be reduced.

Next, a three-dimensional image of the specific observation object is constructed by using the cross-sectional position information and the image data (three-dimensional image generation step S14). Here, for example, a computer (control unit 26) is used to generate (construct) a three-dimensional stereoscopic image of the specific observation object from a plurality of cross-sectional images of the specific portion including the specific observation object by an image processing program.

Since the three-dimensional stereoscopic image of the specific observation object obtained in the stereoscopic image generation step S14 can freely observe the specific observation object from an arbitrary viewpoint and can reproduce an arbitrary cross-sectional image. For example, it becomes possible to observe the internal structure of a cell with SEM resolution.

As a modification of the cross-section processing observation method described above, as shown in the flowchart of FIG. 5, is there a specific observation object for which a three-dimensional stereoscopic image should be constructed after the acquisition of the SEM image of the specific observation object is completed? It is also preferable to provide a step (S15) for determining whether or not to do so.

In the above-described embodiment, the case where there are two specific observation objects (parts of interest) has been described. As a result of optical microscope observation, if it is determined that the specific observation object is only C1, the processing is completed when the cross-sectional processing by the focused ion beam 21 reaches the Z coordinate Z1B. However, when there are a plurality of specific observation objects as shown in FIGS. 3 and 4, the cross-sectional processing by the focused ion beam 21 has reached the time when the processing at narrow intervals is completed (Z coordinate Z1B) according to the flowchart shown in FIG. At the time point), the presence or absence of another specific observation object is then determined (S15). Then, when another specific observation object is present, the cross-section processing step S11 and the cross-section image acquisition step S12 are repeatedly executed. When there is no other specific observation object, a three-dimensional stereoscopic image is generated by the stereoscopic image generation step S14 of the specific observation object.

(Cross-section processing observation method: second embodiment)
In the first embodiment described above, as the simplest example in which a plurality of specific observation objects (parts of interest) exist, an example in which they exist apart from each other and only one specific observation object appears in a certain cross section will be described. However, the present invention is not limited to this.
In the present embodiment, when a plurality of specific observation objects (parts of interest) coexist in the same XY plane (same Z coordinate), the present invention can be applied to the observation of such a sample.
FIG. 8 is a projection drawing (schematic diagram) of a cross section (Z viewpoint) and an XZ plane (Y viewpoint) of the XY plane, which is created from image information obtained by observing the target sample with an optical microscope in advance. Note that, in FIG. 8, the same reference numerals as those in FIG. 2 have the same configuration, and duplicate description will be omitted.

In the present embodiment, the specific observation objects C3 and C4 coexist at the same Z coordinate, and one specific observation object is smaller than the other. When the processed cross section reaches the Z coordinate Z31, the specific observation object C3 appears in the cross section. At this time, similarly to the method described above, the SEM image of the specific portion, which is the observation region defined in advance, is started to be acquired for the specific observation object C3, and the cross-section is processed by the focused ion beam 21 at the predetermined timing and interval. Start.

When the processed cross section reaches the Z coordinate Z41 in the middle of processing and image acquisition for the specific observation object C3, another specific observation object C4 appears. It is known in advance that the specific observation object C4 is an object long in the Z direction by observation with an optical microscope in advance. In the example shown in FIG. 8, the Z coordinate Z41 to the Z coordinate Z32 mean that the specific observation object C3 and the specific observation object C4 are exposed in the same cross section.

The cross-sectional processing interval by the focused ion beam 21 between the Z coordinate Z41 and the Z coordinate Z32 is such that the cross-sectional processing by the focused ion beam 21 is performed at an interval corresponding to the specific observation object C3, but the specific observation object C3 disappears. Even after coordinate Z32, processing is continued up to Z coordinate Z42 at the same processing interval. This is because if the processing interval of the same specific observation object is changed in the middle, a visual discomfort will occur in the finally constructed three-dimensional stereoscopic image. Further, even when comparing the three-dimensional stereoscopic images of the specific observation object C3 and the specific observation object C4, the Z direction can be compared with the same resolution by setting the same processing interval.

In this way, by storing the images of the specific observation object C3 and the specific observation object C4 and the information of the processing interval, the three-dimensional stereoscopic image of the specific observation object C3 and the specific observation object C4 is obtained from these information. Can be built.

(Cross-section processing observation method: Third embodiment)
In the first embodiment described above, an example in which the specific observation object (part of interest) exists along one curve has been described, but the present invention can be applied even when the specific observation object is branched into a plurality of branches. .. It can also be applied when a plurality of specific observation objects are united.
FIG. 9 is a projection drawing (schematic view) of a cross section of the XY plane (viewpoint in the Z direction) and a plane of XZ (viewpoint in the Y direction) obtained by observing the target sample with an optical microscope in advance. Note that, in FIG. 9, the same reference numerals as those in FIG. 2 have the same configuration, and duplicate description will be omitted.

The specific observation object C5 has a shape that branches in the middle like a Y shape, and it is known in advance by observation with an optical microscope that the thickness of each branch is not constant. Here, the branches are C5a, C5b, and C5c, respectively, as shown in FIG.

First, a cross section is started to be formed from the surface of the sample S (Z coordinate Z0) by the focused ion beam 21, and the branch portion C5a of the specific observation object C5 appears at the Z coordinate Z5a1. Processing and observation are continued according to the predetermined processing interval of the focused ion beam 21 and the SEM observation area. When the processed cross section of the sample S reaches the Z coordinate Z5b1, the branch portion C5b of the specific observation object C5 appears.

At this time, the branch portion C5a and the branch portion C5b coexist on the processed cross section. Up to the Z coordinate Z5c1 where the branch C5a and the branch C5b are united, when the branch C5a and the branch C5b coexist in the same cross section, a predetermined observation area (specific) is set according to the size of each specific observation object. The SEM image is acquired according to the site). It is desirable that the processing interval of the focused ion beam 21 is maintained until the specific observation object disappears according to the size of the specific observation object that first appears.

Further, the observation area by SEM may be appropriately changed according to the cross-sectional area of the specific observation object. In the case of FIG. 9, the cross-sectional area of the branch portion C5c gradually decreases between the Z coordinate Z5C1 and the Z coordinate Z52, but the observation area is set each time according to a predetermined standard according to the cross-sectional area of the branch portion C5c. , May be changed. As a result, the amount of image data in the area where the specific observation object does not exist can be reduced. Further, according to the cross-section processing observation method of the present embodiment, even if the shape of the specific observation object is indefinite, the specific observation object in the sample can be three-dimensionally displayed with high resolution in three dimensions. The amount of recorded image data can be reduced. In addition, when a plurality of specific observation objects having different processing conditions and observation conditions are combined, the processing interval is adjusted to the one with the denser processing interval and the one with the higher resolution, respectively. As a result, even when a plurality of objects are united, observation information under desired conditions can be automatically acquired.

(Cross-section processing observation method: Fourth embodiment)
A fourth embodiment of the cross-section processing observation method of the present invention using the cross-section processing observation device shown in FIG. 10 will be described with reference to FIGS. 10 to 14.
11, FIG. 12, FIG. 13, and FIG. 14 are explanatory views showing step by step the observation procedure in the fourth embodiment of the cross-section processing observation method of the present invention.
First, an X-ray CT image of sample S is obtained in an X-ray CT apparatus.

Then, as shown in FIG. 11, by observing the X-ray CT image of the sample S, a specific portion (a specific portion) which is a small region in the sample S including the specific observation object (attention portion) C11 contained in the sample S (the specific portion). The region of interest) Q is determined, and the three-dimensional position coordinate (XYZ coordinate) information thereof is acquired (position information acquisition step).

Next, based on the three-dimensional position coordinate information of the specific part Q, the focused ion beam 21 is irradiated from the focused ion beam barrel 11 toward the specific part Q where the specific observation object C11 exists, and the sample S is subjected to. Perform cross-section forming processing of specific part Q (cross-section processing step). The details of the cross-section forming process of the sample S by the focused ion beam 21 are the same as those in the first embodiment.

Then, among the cross sections of the sample S processed at predetermined intervals, the electron beam (EB) 22 is irradiated from the electron beam (EB) lens barrel 12 toward the specific portion Q including the specific observation object C11, and the sample is sampled. The characteristic X-ray generated from the specific portion Q of S is detected by the EDS detector 51. Then, as shown in FIG. 12, a cross-sectional composition image is constructed for each cross-section of the sample S. At this time, it is possible to obtain a composition image of the entire cross section having the specific portion Q and a high-resolution composition image of a small region including the specific observation object C11.

For example, in the example shown in FIG. 12, three cross sections F1, F2, and F3 are preset in the specific portion Q, and each cross section is processed by the focused ion beam 21. Then, in each of the cross sections F1, F2, and F3, the composition images K1a, K2a, and K3a of the entire cross section and the high resolution cross-sectional composition images K1b, K2b, and K3b of a small region including the specific observation object C11 are acquired. In the example of FIG. 12, in the cross sections F1, F2, and F3 of the specific portion Q set to be rectangular, the composition images K1a, K2a, and K3a of the entire cross section have the specific observation objects (specific observation objects) in the components G1, G2, and G3. Attention part) There is C11. Then, according to the high-resolution cross-sectional composition images K1b, K2b, and K3b, the specific observation object C11 is composed of the components G4, G5, and G6.

In each processed cross section of the specific portion Q, only the cross-sectional composition image of the entire cross section may be acquired, and when there are two or more specific observation objects, two or more high-resolution cross sections are obtained. It is also possible to obtain a composition image. Further, the cross-sectional composition image may be acquired for all the processed cross sections of the specific portion Q, or the cross-sectional composition image may be selectively acquired only for the specific cross section.

By the above steps, for example, as shown in FIG. 13, in the three cross sections F1, F2, F3 of the specific portion Q, the composition images K1a, K2a, K3a of the entire cross section and the small object including the specific observation object C11 are included. High-resolution cross-sectional composition images K1b, K2b, and K3b of the region can be obtained.

Next, FIG. 14 shows the cross-sectional position information of the sample S, the composition images K1a, K2a, and K3a in the respective cross-sections F1, F2, and F3, and the high-resolution cross-sectional composition images K1b, K2b, and K3b. A three-dimensional three-dimensional composition image R of a specific part Q including such a specific observation object is constructed (three-dimensional image generation step). Here, for example, a computer (control unit 26) is used by an image processing program to construct a specific observation object. A three-dimensional three-dimensional composition image of a specific part Q including the above is generated (constructed).

The three-dimensional composition image R obtained by the three-dimensional image generation step is obtained by synthesizing a three-dimensional composition image showing the components of the entire specific part Q and a high-resolution three-dimensional composition image obtained by analyzing a specific observation object with high resolution. Become.

As described above, by acquiring the cross-sectional composition image by detecting the characteristic X-rays in each processed cross section with the EDS detector 51, the three-dimensional three-dimensional composition image R of the specific portion Q including the specific observation object is obtained. be able to.

Although some embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, as well as in the scope of the invention described in the claims and the equivalent scope thereof.

10 Cross-section processing observation device 11 Focused ion beam (FIB) lens barrel 12 Electron beam (EB) lens barrel 13 Optical microscope tube (optical microscope) 13
26 Control 51 EDS detector

Claims (6)

The entire sample to be observed by observing with an optical microscope, a position information acquisition step of acquiring the approximate three-dimensional position coordinate information within said sample of a particular observation object contained in the sample,
Based on the three-dimensional position coordinate information, a cross-section processing step of irradiating a specific portion of the sample in which the specific observation object is present with a focused ion beam to expose the cross section of the specific portion.
A cross-sectional image acquisition step of irradiating the cross-section with an electron beam to acquire an image of a region of a predetermined size including the specific observation object, and a cross-sectional image acquisition step.
The cross section processing step and the cross-sectional image acquisition process repeated several times along a predetermined direction at a predetermined interval, generating stereoscopic images to construct a three-dimensional image including the specific observation object from a plurality of pre-outs image acquired A method for observing cross-section processing, which comprises a process. The cross-section processing observation method according to claim 1, wherein a plurality of types of the specific observation object are set, and the cross-section processing step is performed for each specific observation object. The cross-section processing observation method according to claim 1 or 2, wherein a confocal stereomicroscope is used as the optical microscope. In the cross-sectional image acquisition step, a cross-sectional composition image of the specific portion including the specific observation object is further acquired by energy dispersion type X-ray detection of the cross section.
The cross-section processing observation method according to claim 1 or 2, wherein a three-dimensional three-dimensional composition image including the specific observation object is constructed from the plurality of acquired cross-section composition images in the three-dimensional image generation step. A sample table on which a sample containing a specific observation object is placed, and
A focused ion beam lens barrel that irradiates the sample with a focused ion beam,
An electron beam lens barrel that irradiates the sample with an electron beam,
An optical microscope tube for optically observing the sample and
A secondary electron detector that detects secondary electrons generated from the sample or a backscattered electron detector that detects backscattered electrons,
The position of the specific observation object in the sample is specified by the optical microscope cylinder, and the focused ion beam is emitted from the focused ion beam lens barrel toward the specific part of the sample in which the specific observation object is present. The cross section is exposed by irradiation, and the cross section is irradiated with an electron beam from the electron beam lens barrel to acquire an image of a region of a predetermined size including the specific observation object, and the specific observation object is obtained from the image . A control unit that constructs a three-dimensional stereoscopic image of a specific part including
A cross-section processing observation device characterized by being equipped with. The cross-section processing observation device according to claim 5, further comprising an EDS detector that detects characteristic X-rays generated from the sample, and the control unit constructs a three-dimensional three-dimensional composition image of the specific portion.

Images