Scanning electron microscope

Abstract

To provide a scanning electron microscope capable of reducing positional deviation of a photographing position.SOLUTION: A scanning electron microscope for performing three dimensional reconstruction by imaging each section constituting a continuous section in which a plurality of sections continuously cut out from a sample are connected, the scanning electron microscope including a control unit that performs a process of moving a sample stage to a first imaging position of a first section, a process of capturing a first positioning image at the first imaging position, a process of moving the sample stage to a second imaging position of a second section, and a process of: A process of capturing a second positioning image, a process of obtaining a first deviation amount between the first positioning image and the second positioning image and determining whether or not the first deviation amount is smaller than a first threshold value, and a process of correcting the second imaging position such that the first deviation amount is reduced in a case where it is determined that the first deviation amount is not smaller than the first threshold value.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present invention relates to a scanning electron microscope. [Background technology]

[0002] One method for analyzing the three-dimensional structure of biological samples is array tomography, in which serial sections are prepared from resin-embedded biological samples using a microtome or other device, the same location on each section is photographed using an electron microscope, and three-dimensional reconstruction is performed from multiple consecutive cross-sectional images.

[0003] For example, Patent Document 1 discloses a pretreatment method for introducing serial sections cut from a biological sample into an electron microscope. A plurality of consecutive cross-sectional images obtained by photographing each of the sections constituting the serial section with an electron microscope are aligned by image processing. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-106483 Summary of the Invention [Problem to be solved by the invention]

[0005] However, if there is a large misalignment between consecutive cross-sectional images, the area wasted due to the alignment of the images will be large. [Means for solving the problem]

[0006] One aspect of the scanning electron microscope according to the present invention is A scanning electron microscope for photographing and reconstructing three-dimensional images of a series of consecutive sections cut out from a sample, a sample stage for positioning the serial sections; an electron optical system that scans each of the slices with an electron beam; a detector for detecting electrons emitted by scanning each of the slices with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit a process of moving the sample stage to a first imaging position of a first slice; A process of capturing a first alignment image at the first imaging position; a process of moving the sample stage to a second imaging position of a second slice; A process of capturing a second alignment image at the second photographing position; a process of calculating a first amount of deviation between the first alignment image and the second alignment image, and determining whether the first amount of deviation is smaller than a first threshold value; a process of correcting the second photographing position so as to reduce the first deviation amount when it is determined that the first deviation amount is not smaller than the first threshold value; A process of capturing a third alignment image at the corrected second imaging position; a process of calculating a second amount of deviation between the first alignment image and the third alignment image, and determining whether the second amount of deviation is smaller than the first threshold value; Do the following.

[0007] In such a scanning electron microscope, the positional deviation between the first photographing position of the first slice and the second photographing position of the second slice can be reduced, thereby reducing the positional deviation between the image photographed at the first photographing position and the image photographed at the second photographing position.

[0008] One aspect of the scanning electron microscope according to the present invention is A scanning electron microscope for photographing and reconstructing three-dimensional images of a series of consecutive sections cut out from a sample, a sample stage for positioning the serial sections; an electron optical system that scans the slice with an electron beam; a detector for detecting electrons emitted by scanning the slice with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit A process of capturing a first alignment image at a first capturing position of a first slice; A process of capturing a second alignment image at a second photographing position different from the first photographing position of the first slice; A process of calculating a first deviation amount between the first alignment image and the second alignment image; a process of determining whether a first difference between the amount of movement from the first imaging position to the second imaging position and the first amount of displacement is smaller than a first threshold value; a process of correcting the second photographing position so as to reduce the first difference when it is determined that the first difference is not smaller than the first threshold value; A process of capturing a third alignment image at the corrected second imaging position; A process of calculating a second deviation amount between the first alignment image and the third alignment image; a process of determining whether a second difference between the movement amount and the second deviation amount is smaller than the first threshold value; a process of correcting the second shooting position so as to reduce the second difference when it is determined that the second difference is not smaller than the first threshold value; Do the following.

[0009] In such a scanning electron microscope, the second photographing position can be accurately positioned relative to the first photographing position of the first section.

[0010] One aspect of the scanning electron microscope according to the present invention is a sample stage for positioning the sample; an electron optical system that scans the sample with an electron beam; a detector for detecting electrons emitted by scanning the sample with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit A process of capturing a first target image at a first imaging position and a first magnification; a process of capturing a first alignment image at a second magnification lower than the first magnification; a process of capturing a second alignment image at a third magnification that is lower than the second magnification; Processing to switch shooting conditions, a process of capturing a third alignment image at the third magnification; a process of calculating a first deviation amount between the second alignment image and the third alignment image, and determining whether the first deviation amount is smaller than a first threshold value; a process of correcting the first photographing position so as to reduce the first deviation amount when it is determined that the first deviation amount is not smaller than the first threshold value; a process of capturing a fourth alignment image at the second magnification when it is determined that the first deviation amount is smaller than the first threshold value; A second deviation amount between the first alignment image and the fourth alignment image is calculated. a process of determining whether the second deviation amount is smaller than a second threshold value; a process of correcting the first photographing position so as to reduce the second deviation amount when it is determined that the second deviation amount is not smaller than the second threshold value; a process of capturing a second target image at the first magnification when it is determined that the second deviation amount is smaller than the second threshold value; Do the following.

[0011] In such a scanning electron microscope, the shooting position is corrected using the second alignment image and the third alignment image, and then the shooting position is corrected using the first alignment image and the fourth alignment image, so the shooting position can be corrected accurately. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an example of the configuration of an electron microscope according to a first embodiment. [Figure 2] FIG. 2 is a diagram showing an example of the configuration of a control unit. [Figure 3]FIG. 1 is a diagram illustrating a method for preparing serial sections. [Figure 4] FIG. 2 is a diagram showing a schematic diagram of serial sections placed on a sample stage. [Figure 5] 10 is a flowchart showing an example of a three-dimensional reconstruction process. [Figure 6] FIG. 3 is a diagram for explaining each step of the 3D reconstruction process. [Figure 7] FIG. 3 is a diagram for explaining each step of the 3D reconstruction process. [Figure 8] FIG. 3 is a diagram for explaining each step of the 3D reconstruction process. [Figure 9] Figures showing examples of high-magnification images and three-dimensional images of each section. [Figure 10] 10 is a flowchart showing an example of high-magnification image capturing processing. [Figure 11] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 12] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 13] 10 is a flowchart showing an example of high-magnification image capturing processing. [Figure 14] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 15] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 16] 10 is a flowchart showing a modified example of high-magnification image capturing processing. [Figure 17] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 18] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 19] 10 is a flowchart showing an example of high-magnification image capturing processing. [Figure 20] 10 is a flowchart showing an example of high-magnification image capturing processing. [Figure 21] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 22] 10 is a flowchart showing an example of high-magnification image capturing processing. [Figure 23] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. [Figure 24] 5A to 5C are diagrams for explaining each step of the high-magnification image capturing process. DETAILED DESCRIPTION OF THE INVENTION

[0013] Preferred embodiments of the present invention will be described in detail below with reference to the drawings. Note that the embodiments described below do not unduly limit the content of the present invention as defined in the claims. Furthermore, not all of the configurations described below are necessarily essential components of the present invention.

[0014] 1. First embodiment 1.1. Electron microscope First, an electron microscope according to the first embodiment will be described with reference to the drawings. Fig. 1 is a diagram showing an example of the configuration of an electron microscope 100 according to the first embodiment.

[0015] The electron microscope 100 is a scanning electron microscope that scans a sample with an electron beam to obtain a scanning electron microscope image (SEM image). As shown in FIG. 1, the electron microscope 100 includes an electron optical system 10, a sample stage 20, a detector 30, and a control unit 40.

[0016] The electron optical system 10 irradiates an electron beam onto a sample S supported on a sample stage 20. The electron optical system 10 is composed of an electron gun, a focusing lens, an objective lens, an astigmatism corrector, a deflector, etc. In the electron optical system 10, the electron beam emitted from the electron gun is focused by the focusing lens and the objective lens to form an electron probe, and the electron beam is deflected by the deflector to scan the sample S with the electron probe.

[0017] The detector 30 detects electrons (secondary electrons) emitted from the sample S by irradiating the sample S with an electron beam. The detector 30 may be a detector that detects backscattered electrons emitted from the sample S by irradiating the sample S with an electron beam.

[0018] In the electron microscope 100, an electron optical system 10 forms an electron probe, which is used to scan a sample S. A detector 30 detects electrons emitted from the sample S in synchronization with the scanning of the electron probe, thereby capturing an SEM image (a secondary electron image or a backscattered electron image).

[0019] The control unit 40 controls each unit of the electron microscope 100. Fig. 2 is a diagram showing an example of the configuration of the control unit 40. As shown in Fig. 2, the control unit 40 includes a processing unit 42, an operation unit 44, a display unit 46, and a storage unit 48.

[0020] The operation unit 44 is used by the user to input operation information, and outputs the input operation information to the processing unit 42. The functions of the operation unit 44 can be realized by input devices such as a keyboard, a mouse, buttons, a touch panel, and a touch pad.

[0021] The display unit 46 displays the image generated by the processing unit 42. The function of the display unit 46 can be realized by an LCD (Liquid Crystal Display), a touch panel display, or the like.

[0022] The storage unit 48 stores programs, data, etc. for the processing unit 42 to perform various calculation processes and various control processes. The storage unit 48 is also used as a working area for the processing unit 42, and is also used to temporarily store the results of calculations performed by the processing unit 42 in accordance with the various programs. The functions of the storage unit 48 can be realized by a RAM (Random Access Memory), a ROM (Read Only Memory), a hard disk, etc.

[0023] The functions of the processing unit 42 can be realized by various processors such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), and a DSP (Digital Signal Processor) by executing programs stored in the storage unit 48. The processing unit 42 includes a stage control unit 420, an imaging unit 422, and an image processing unit 424.

[0024] The stage control unit 420 controls the sample stage 20. The photographing unit 422 performs processing to photograph an SEM image of the sample S. Specifically, the photographing unit 422 acquires information on the intensity of electrons detected by the detector 30 while causing the electron optical system 10 to scan the sample S with an electron probe. This makes it possible to acquire an SEM image. The image processing unit 424 performs processing such as calculating the amount of positional deviation between two SEM images and performing three-dimensional reconstruction. Details of the processing by the stage control unit 420, the photographing unit 422, and the image processing unit 424 will be described later.

[0025] 1.2. 3D reconstruction Array tomography is a technique in which a series of consecutive sections, each of which is cut out successively by a microtome, is photographed to obtain multiple cross-sectional images, and then 3D reconstruction is performed using the obtained multiple cross-sectional images. The electron microscope 100 can photograph high-magnification images of each of the consecutive sections to perform 3D reconstruction.

[0026] Fig. 3 is a diagram for explaining a method for preparing serial sections 4. Fig. 3 illustrates three axes that are perpendicular to one another: an X axis, a Y axis, and a Z axis.

[0027] As shown in Figure 3, a biological sample 2 is cut using a microtome or the like to continuously cut out slices 6. This allows the production of serial slices 4, which are a series of slices 6. In the example shown in Figure 3, slices 6 of the XY cross section of the sample S can be cut out by moving the microtome blade along the Z axis.

[0028] FIG. 4 is a schematic diagram showing a state in which a serial section 4 is placed on a sample stage 20. As shown in FIG. 4, the serial section 4 is supported on a substrate 3. The serial section 4 is placed on the sample stage 20 while being supported on the substrate 3. The substrate 3 is, for example, a semiconductor substrate such as a silicon substrate. In the example shown in FIG. 4, the serial section 4 is made up of a first section 6-1, a second section 6-2, a third section 6-3, ..., an Nth section 6-N (N is an integer of 2 or more).

[0029] Processing 1.3.1. 3D reconstruction processing Fig. 5 is a flowchart showing an example of 3D reconstruction processing in the electron microscope 100. Here, a case will be described in which N slices 6 are connected to form a continuous section 4. Figs. 6 to 8 are diagrams for explaining each step of the 3D reconstruction processing.

[0030] First, the photographing unit 422 photographs a low-magnification image I1 including the entire serial section 4, as shown in FIG. 6 (step S10).

[0031] The photographing unit 422, for example, links the coordinates with an optical microscope image of the serial section 4, and then photographs an SEM image at low magnification that includes the entire serial section 4. This makes it possible to obtain a low-magnification image I1.

[0032] Next, the imaging unit 422 sets imaging positions P2-1 to P2-N of the medium-magnification images I2-1 to I2-N of each slice 6 on the low-magnification image I1 as shown in FIG. 7 (step S12).

[0033] The imaging unit 422 sets imaging positions P2-1 to P2-N so that the same location of the biological sample 2 is imaged on each slice 6. For example, the imaging unit 422 sets the position specified by the user on the first slice 6-1 as imaging position P2-1, and sets imaging positions on the other slices 6 so that the same location of the biological sample 2 as the location imaged in the medium-magnification image I2-1 of the first slice 6-1 is imaged. Here, for example, the same location of the biological sample 2 on the first slice 6-1 and the second slice 6-2 is a location where the coordinates (X, Y) in the slice 6-1 and the coordinates (X, Y) in the slice 6-2 are the same in the coordinate system of the biological sample 2 shown in FIG. 3.

[0034] Next, the stage control unit 420 and the photographing unit 422 photograph medium-magnification images I2-1 to I2-N of each slice 6 (step S14).

[0035] The stage control unit 420 moves the sample stage 20 to the imaging position P2-1 of the first slice 6-1 set in step S12. The imaging position can be changed by moving the sample stage 20. Next, the imaging unit 422 captures an SEM image at the imaging position P2-1. This allows the medium-magnification image I2-1 of the first slice 6-1 shown in FIG. 7 to be captured.

[0036] Next, the stage control unit 420 moves the sample stage 20 to the photographing position P2-2 of the second slice 6-2, and the photographing unit 422 photographs an SEM image at the photographing position P2-2. This allows a medium-magnification image I2-2 of the second slice 6-2 to be captured. The same process is performed for the third and subsequent slices 6, and medium-magnification images I2-1 to I2-N of each slice 6 are captured.

[0037] Next, the image processing unit 424 corrects the photographing positions P2-1 to P2-N of the medium-magnification images I2-1 to I2-N of each slice 6 (step S16).

[0038] The image processing unit 424 extracts feature points between the medium-magnification image I2-1 of the first slice 6-1 and the medium-magnification image I2-2 of the second slice 6-2, and calculates the amount of positional shift between the two images by matching the extracted feature points. Based on this amount of positional shift, the image processing unit 424 corrects the imaging position P2-2 of the medium-magnification image I2-2 so that the area of ​​the biological sample 2 captured in the medium-magnification image I2-1 and the area of ​​the biological sample 2 captured in the medium-magnification image I2-2 are the same area. The method for calculating the amount of positional shift between images is not particularly limited, and any known method can be used.

[0039] Similarly, the image processing unit 424 calculates the amount of positional deviation between the medium-magnification image I2-2 of the second slice 6-2 and the medium-magnification image I2-3 of the third slice 6-3, and corrects the imaging position P2-3 of the third slice 6-3. Similar processing is performed for the medium-magnification images of the third and subsequent slices 6, and corrects the imaging positions P2-1 to P2-N of the medium-magnification images I2-1 to I2-N of each slice 6.

[0040] Next, the processing unit 42 photographs a high-magnification image of each slice 6 (step S18), as shown in Fig. 8. The process S18 for photographing high-magnification images I4-1 to I4-N will be described later. By the process S18 for photographing high-magnification images, high-magnification images I4-1, I4-2, ..., I4-N of the same location on the biological sample 2 can be photographed.

[0041] Next, the image processing unit 424 performs three-dimensional reconstruction of the biological sample 2 using the high-magnification images I4-1, I4-2, . . . , I4-N of each slice 6 (step S20).

[0042] The image processing unit 424 aligns the high-magnification images I4-1, I4-2, ..., I4-N of each slice 6 and stacks the aligned high-magnification images I4-1, I4-2, ..., I4-N of each slice 6 to 3D reconstruct the biological sample 2. The image processing unit 424 displays the 3D image obtained by 3D reconstruction on the display unit 46, and then terminates the 3D reconstruction process. Note that the method for 3D reconstruction from the high-magnification images I4-1, I4-2, ..., I4-N of each slice 6 is not particularly limited, and any known method can be used.

[0043] 9 is a diagram showing an example of a high-magnification image and a three-dimensional image I6 of each slice 6. As shown in FIG. 8, a three-dimensional image I6 of the biological sample 2 can be generated by aligning the high-magnification images I4-1, I4-2, . . . , I4-N of each slice 6 and stacking the aligned high-magnification images I4-1, I4-2, . . . , I4-N. The electron microscope 100 can accurately align the high-magnification images I4-1, I4-2, . . . , I4-N, and therefore can generate a good three-dimensional image I6.

[0044] 1.3.2. High-magnification image capture processing Fig. 10 is a flowchart showing an example of the high-magnification image capturing process S18 in the electron microscope 100. Fig. 11 and Fig. 12 are diagrams for explaining each step of the high-magnification image capturing process.

[0045] First, the stage control unit 420 moves the sample stage 20 to an imaging position where a high-magnification image of the Mth slice 6 is captured (step S100). Here, M=1, and the stage control unit 420 moves the sample stage 20 to an imaging position P4-1 where a high-magnification image I4-1 of the first slice 6-1 is captured.

[0046] The photographing position P4-1 for capturing the high-magnification image I4-1 can be specified by the user on the medium-magnification image I2-1 of the first slice 6-1, as shown in FIG. 11. Once the photographing position P4-1 for capturing the high-magnification image I4-1 on the first slice 6-1 is set, the photographing unit 422 sets the photographing position P4-2 on the second slice 6-2 so that the same location of the biological sample 2 captured in the high-magnification image I4-1 is captured. For example, the photographing unit 422 sets the photographing position P4-2 based on the positional relationship (distance and direction) between the photographing positions P4-1 and P2-1, using the photographing position P2-2 as a reference. The photographing unit 422 also sets the photographing positions P2-3 to P2-N on the third and subsequent slices 6 so that the same location of the biological sample 2 captured in the high-magnification image I4-1 is captured.

[0047] Next, the photographing unit 422 photographs an alignment image I10-1 at the photographing position P4-1 (step S102). The alignment image I10-1 is an image for correcting the photographing position P4-1. The magnification of the alignment image I10-1 is different from the magnification of the high-magnification image I4-1.

[0048] The alignment image I10-1 is set to imaging conditions that allow accurate alignment between images. For example, the magnification of the alignment image I10-1 can be set according to the expected amount of misalignment between images. If the expected amount of misalignment between images is on the order of several microns, accurate correction is possible if the width of the field of view is on the order of several tens of microns. Therefore, if the expected amount of misalignment between images is on the order of several microns, the magnification of the alignment image I10-1 is set to a magnification that results in a width of the field of view of on the order of several tens of microns. The magnification of the alignment image I10-1 may also be set based on the required accuracy of alignment between images.

[0049] As shown in FIG. 11, the magnification of the alignment image I10-1 may be lower than that of the high-magnification image I4-1. For example, when aligning high-magnification SEM images, which have a narrow field of view and unclear contours of features, the field of view may be lost, making alignment impossible. Compared to high-magnification SEM images, low-magnification SEM images often contain more features within the field of view and have clearer contours of the features. Therefore, by performing alignment using the low-magnification alignment image I10-1, the possibility of losing the field of view is reduced compared to when performing alignment using the high-magnification image I4-1.

[0050] Next, the photographing unit 422 photographs a high-magnification image I4-1 at a photographing position P4-1 as shown in FIG. 11 (step S104).

[0051] Next, the stage control unit 420 moves the sample stage 20 to an imaging position where a high-magnification image of the (M+1)th slice 6 is captured (step S106). Here, M=1, and the stage control unit 420 moves the sample stage 20 to an imaging position P4-2 where a high-magnification image I4-2 of the second slice 6-2 is captured, as shown in FIG.

[0052] Next, the photographing unit 422 photographs the alignment image I10-2 at the photographing position P4-2 (step S108). The photographing conditions for the alignment image I10-2 are the same as the photographing conditions for the alignment image I10-1.

[0053] Next, the image processing unit 424 calculates the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 (step S110).

[0054] The image processing unit 424 extracts feature points between the alignment images I10-1 and I10-2, and calculates the amount of misalignment between the two images by matching the extracted feature points. The method for calculating the amount of misalignment between the images is not particularly limited, and a known method may be used. The amount of misalignment can be expressed as a misalignment amount dx1 in the X direction, a misalignment amount dy1 in the Y direction, and a misalignment amount dθ1 in the rotation direction.

[0055] Next, the image processing unit 424 determines whether the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 is smaller than a threshold value (step S112).

[0056] The image processing unit 424 compares the X-direction misalignment amount dx1 between the alignment images I10-1 and I10-2 with an X-direction misalignment threshold Tx1 to determine whether the misalignment amount dx1 is smaller than the threshold Tx1. Similarly, the image processing unit 424 compares the Y-direction misalignment amount dy1 between the alignment images I10-1 and I10-2 with a Y-direction misalignment threshold Ty1 to determine whether the misalignment amount dy1 is smaller than the threshold Ty1. Similarly, the image processing unit 424 compares the rotational direction misalignment amount dθ1 between the alignment images I10-1 and I10-2 with a rotational direction misalignment threshold Tθ1 to determine whether the misalignment amount dθ1 is smaller than the threshold Tθ1.

[0057] The thresholds Tx1, Ty1, and Tθ1 can be set appropriately depending on the required alignment accuracy. For example, a GUI (graphical user interface) screen for setting the thresholds Tx1, Ty1, and Tθ1 is displayed on the display unit 46, and the user can set the thresholds Tx1, Ty1, and Tθ1 via the GUI screen. Note that the user may input an index representing the alignment accuracy, and the thresholds Tx1, Ty1, and Tθ1 may be set according to this index. Note that the same applies to other thresholds described below.

[0058] If any one of the positional deviation amounts dx1, dy1, and dθ1 is not smaller than the threshold, the image processing unit 424 determines that the positional deviation amount between the positional deviation images I10-1 and I10-2 is not smaller than the threshold. On the other hand, if all of the positional deviation amounts dx1, dy1, and dθ1 are smaller than the threshold, the image processing unit 424 determines that the positional deviation amount between the positional deviation images I10-1 and I10-2 is smaller than the threshold.

[0059] If the image processing unit 424 determines that the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 is not smaller than the threshold value (No in step S112), the stage control unit 420 corrects the shooting position P4-2 as shown in FIG. 12 (step S114).

[0060] The stage controller 420 corrects the imaging position P4-2 by moving the sample stage 20 so that the positional deviation amounts dx1, dy1, and dθ1 become smaller. The stage controller 420 corrects the imaging position P4-2 by moving or rotating the sample stage 20 in the horizontal direction. Note that the stage controller 420 may correct the imaging position P4-2 by moving the imaging position, for example, by image shift, which deflects the electron beam to move the field of view, or by scan rotation, which changes the scanning direction of the electron beam to rotate the SEM image. In this way, the imaging position P4-2 may be corrected by moving the sample stage 20, or by moving the imaging position using the electron optical system 10.

[0061] After the stage control unit 420 corrects the photographing position P4-2 (after step S114), the process returns to step S108, and the photographing unit 422 photographs the alignment image I10-2 at the corrected photographing position P4-2. The image processing unit 424 calculates the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 (step S110), and It is determined whether the amount of positional deviation is smaller than a threshold value (step S112).

[0062] In this way, the processing unit 42 repeats steps S108 to S114 until it is determined that the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 is smaller than the threshold value.

[0063] If the image processing unit 424 determines that the amount of positional deviation between the alignment image I10-1 and the alignment image I10-2 is smaller than the threshold value (Yes in step S112), the photographing unit 422 photographs a high-magnification image I4-2 at the photographing position P4-2 (step S116), as shown in Fig. 12. The photographing conditions for the high-magnification image I4-2 are the same as those for the high-magnification image I4-1.

[0064] Next, the photographing unit 422 determines whether high-magnification images have been photographed for all slices 6, i.e., whether M=N (step S118). If the photographing unit 422 determines that high-magnification images have not been photographed for all slices 6, i.e., that M=N is not true (No in step S118), it sets M=M+1 (step S120) and returns to step S106, where it performs process S106 of moving the sample stage 20 to the photographing position for the (M+1)-th slice 6. Here, in step S120, M=2, and in process S106, the stage control unit 420 moves the sample stage 20 to the photographing position P4-3 for the third slice 6-3.

[0065] The photographing unit 422 photographs the alignment image I10-3 at the photographing position P4-3 (step S108), and the image processing unit 424 calculates the amount of misalignment between the alignment image I10-2 and the alignment image I10-3 (step S110), and the image processing unit 424 determines whether the amount of misalignment is smaller than a threshold (step S112). If the image processing unit 424 determines that the amount of misalignment is not smaller than the threshold (No in step S112), the stage control unit 420 corrects the photographing position P4-3 (step S114), and the process returns to step S108, where the photographing unit 422 photographs the alignment image I10-3 at the corrected photographing position P4-3 (step S108). The processing unit 42 repeats steps S108 to S114 until the amount of misalignment between the alignment image I10-2 and the alignment image I10-3 becomes smaller than the threshold. If the image processing unit 424 determines that the amount of positional deviation is smaller than the threshold value (Yes in step S112), the photographing unit 422 photographs a high-magnification image I4-3 at the photographing position P4-3 (step S116).

[0066] The processing unit 42 also performs steps S106 to S116 on the fourth and subsequent slices 6, capturing high-magnification images I4-3 to I4-N of each slice 6. In this manner, the processing unit 42 repeats steps S106 to S120 and captures high-magnification images I4-1 to I4-N of each slice 6 until it is determined that M=N.

[0067] When the photographing unit 422 determines that high-magnification images have been photographed for all slices 6, that is, that M=N is satisfied (Yes in step S118), the high-magnification image photographing process S18 ends.

[0068] Effects The electron microscope 100 according to the first embodiment is a scanning electron microscope for photographing and 3D reconstructing each of the slices 6 that make up a serial section 4, which is a series of multiple slices 6 cut consecutively from a biological sample 2, and includes a sample stage 20 that positions the serial section 4, an electron optical system 10 that scans each of the slices 6 with an electron beam, a detector 30 that detects electrons emitted by scanning each of the slices 6 with the electron beam, and a control unit 40 that controls the sample stage 20 and the electron optical system 10.

[0069] The control unit 40 also performs the following processes: moving the sample stage 20 to an imaging position P4-1 (an example of a first imaging position) of the first slice 6-1 (an example of a first slice), and capturing an alignment image I10-1 (an example of a first alignment image) at the imaging position P4-1. The control unit 40 also performs the following processes: moving the sample stage 20 to an imaging position P4-2 (an example of a second imaging position) of the second slice 6-2 (an example of a second slice), and capturing an alignment image I10-2 (an example of a second alignment image) at the imaging position P4-2.

[0070] The control unit 40 also performs the following processes: determining the amount of misalignment between the alignment image I10-1 and the alignment image I10-2, and determining whether the amount of misalignment is smaller than a threshold value; if it determines that the amount of misalignment is not smaller than the threshold value, correcting the shooting position P4-2 so that the amount of misalignment is smaller; photographing the alignment image I10-2 (an example of a third alignment image) at the corrected shooting position P4-2; and determining the amount of misalignment between the alignment image I10-1 and the alignment image I10-2, and determining whether the amount of misalignment is smaller than a threshold value.

[0071] In this way, in the electron microscope 100, the positional deviation between the photographing position P4-1 of the first slice 6-1 and the photographing position P4-2 of the second slice 6-2 can be reduced, and therefore the positional deviation between the high-magnification image I4-1 photographed at the photographing position P4-1 and the high-magnification image I4-2 photographed at the photographing position P4-2 can be reduced.

[0072] Therefore, with the electron microscope 100, for example, there is no need to increase the field of view for capturing high-magnification images to take into account positional deviations, and therefore the capture time can be shortened. Furthermore, with the electron microscope 100, there is no need to increase the field of view for capturing high-magnification images, and therefore the image size of the high-magnification images can be reduced. In particular, when the number of slices 6 reaches several thousand and a large number of high-magnification images are captured, the effect of shortening the capture time and reducing the image size is significant. Furthermore, with the electron microscope 100, positional deviations in the capture position of high-magnification images can be reduced, and therefore the accuracy of alignment during 3D reconstruction can be improved.

[0073] In the electron microscope 100, the control unit 40 performs the following processes: capturing a high-magnification image I4-1 (an example of a first target image) at a magnification different from that of the alignment image I10-1 at the imaging position P4-1; and, if it is determined that the amount of misalignment is smaller than the threshold, capturing a high-magnification image I4-2 (an example of a second target image) at a magnification different from that of the alignment image I10-2 at the imaging position P4-2. Therefore, in the electron microscope 100, alignment between images can be performed using an alignment image at a magnification different from that of the high-magnification image, which is the target image. Therefore, the magnification of the alignment image can be set to a magnification suitable for aligning between images.

[0074] In the electron microscope 100, the control unit 40 sets the magnification of the alignment image I10-1 to be lower than the magnification of the high-magnification image I4-1 in the process of capturing the alignment image I10-1. Therefore, in the electron microscope 100, the possibility of losing sight of the field of view can be reduced compared to when alignment is performed using, for example, the high-magnification image I4-1.

[0075] 2. Second embodiment 2.1. Electron microscope Next, an explanation will be given of an electron microscope according to a second embodiment. The configuration of the electron microscope according to the second embodiment is the same as the configuration of the electron microscope shown in Fig. 1 described above, and therefore the explanation thereof will be omitted.

[0076] Processing In the electron microscope 100 according to the second embodiment, the electron microscope 1 according to the first embodiment described above is 00 differs from the example in the high-magnification image capturing process S18 of the 3D reconstruction process shown in Fig. 5. Below, differences from the example of the electron microscope 100 according to the first embodiment described above will be explained, and explanations of similarities will be omitted.

[0077] Fig. 13 is a flowchart showing an example of the high-magnification image capturing process S18 in the electron microscope 100. Fig. 14 and Fig. 15 are diagrams for explaining each step of the high-magnification image capturing process.

[0078] First, the stage control unit 420 moves the sample stage 20 to the imaging position for the medium-magnification image of the Mth slice 6 (step S200). Here, M=1, and the stage control unit 420 moves the sample stage 20 to the imaging position P2-1 for the medium-magnification image I2-1 of the first slice 6-1, as shown in FIG. 14. Note that the imaging position P2-1 for the medium-magnification image I2-1 has been corrected in process S16 for correcting the imaging positions P2-1 to P2-N shown in FIG. 5. Therefore, as will be described below, the imaging position P4-1 for the high-magnification image I4-1 can be corrected based on the imaging position P2-1.

[0079] The stage control unit 420 moves the sample stage 20 from the imaging position P2-1 to the imaging position P4-1 of the high magnification image I4-1 (step S202). Note that the stage control unit 420 may also move the imaging position from the imaging position P2-1 to the imaging position P4-1 by image shifting.

[0080] Next, the photographing unit 422 photographs an alignment image I20-1 at a photographing position P4-1 (step S204) as shown in Fig. 14. The photographing conditions for the alignment image I20-1 are the same as the photographing conditions for the medium-magnification image I2-1.

[0081] Next, the image processing unit 424 calculates the amount of misalignment between the alignment image I20-1 and the medium-magnification image I2-1 (step S206). The image processing unit 424 extracts feature points between the alignment image I20-1 and the medium-magnification image I2-1, and calculates the amount of misalignment (dx1, dy1, dθ1) between the two images by matching the extracted feature points.

[0082] Next, the image processing unit 424 calculates the difference D between the amount of movement V1 of the photographing position from the photographing position P2-1 to the photographing position P4-1 and the positional deviation amount (dx1, dy1, dθ1) calculated in the process S206 (step S208).

[0083] The movement amount V1 is expressed by the movement amount dx0 in the X direction, the movement amount dy0 in the Y direction, and the movement amount dθ0 in the rotation direction. In the example shown in Fig. 14, the movement of the shooting position from shooting position P2-1 to shooting position P4-1 does not involve rotation, so the movement amount in the rotation direction dθ0 = 0°.

[0084] The image processing unit 424 calculates the difference D between the movement amount V1 (dx0, dy0, dθ0) and the positional deviation amount (dx1, dy1, dθ1) as follows: |dx0-dx1|, the difference between the movement amount dx0 in the X direction and the positional deviation amount dx1 in the X direction; |dy0-dy1|, the difference between the movement amount dy0 in the Y direction and the positional deviation amount dy1 in the Y direction; and |dθ0-dθ1|, the difference between the movement amount dθ0 in the rotational direction and the deviation amount dθ1 in the rotational direction.

[0085] The image processing unit 424 determines whether the difference D is smaller than a threshold value (step S210).

[0086] The image processing unit 424 determines whether the difference |dx0-dx1| in the X direction is smaller than a threshold value Tx2 for the difference in the X direction. Similarly, the image processing unit 424 determines whether the difference |dy0-dy1| in the Y direction is smaller than a threshold value Tx2 for the difference in the Y direction. is smaller than the threshold value Ty2 for the difference in the Y direction. Similarly, the image processing unit 424 determines whether the difference in the rotational direction |dθ0-dθ1| is smaller than the threshold value Ty2 for the difference in the rotational direction. The methods for setting the threshold values ​​Tx2, Ty2, and Tθ2 are the same as the methods for setting the threshold values ​​Tx1, Ty1, and Tθ1 described above, and detailed description thereof will be omitted.

[0087] The image processing unit 424 determines that the difference D is not smaller than the threshold value when any one of the differences |dx0-dx1|, |dy0-dy1|, and |dθ0-dθ1| is not smaller than the threshold value. On the other hand, the image processing unit 424 determines that the difference D is smaller than the threshold value when all of the differences |dx0-dx1|, |dy0-dy1|, and |dθ0-dθ1| are smaller than the threshold value.

[0088] If the image processing unit 424 determines that the difference D is not smaller than the threshold value (No in step S210), the stage control unit 420 corrects the shooting position P4-1 so that the differences |dx0-dx1|, |dy0-dy1|, and |dθ0-dθ1| become smaller (step S212). The process S212 for correcting the shooting position P4-1 is performed in the same manner as the process S114 for correcting the shooting position P4-2 in FIG. 10 described above, and therefore a detailed description thereof will be omitted.

[0089] After the stage control unit 420 corrects the photographing position P4-1 (after step S212), the process returns to step S204, where the photographing unit 422 photographs the alignment image I20-1 at the corrected photographing position P4-1 (step S204). The image processing unit 424 calculates the amount of positional deviation between the alignment image I20-1 and the medium-magnification image I2-1 (step S206), calculates the difference D between the movement amount V1 and the amount of positional deviation (step S208), and determines whether the difference D is smaller than a threshold value (step S210). In this way, the processing unit 42 repeats steps S204 to S212 until it is determined that the difference D is smaller than the threshold value.

[0090] If the image processing unit 424 determines that the difference D is smaller than the threshold value (Yes in step S210), the photographing unit 422 photographs a high-magnification image I4-1 at the photographing position P4-1 as shown in FIG. 14 (step S214).

[0091] The photographing unit 422 determines whether M=N for all slices 6 (step S216). If the photographing unit 422 determines that M=N is not true (No in step S216), it sets M=M+1 (step S218) and returns to step S200 to perform process S200 of moving the sample stage 20 to the photographing position for the (M+1)th slice 6. Here, in step S218, M=2, and in process S200, the stage control unit 420 moves the sample stage 20 to photographing position P2-2 for the medium-magnification image I2-2 of the second slice 6-2.

[0092] The high-magnification image I4-2 of the second slice 6-2 is captured in the same manner as the high-magnification image I4-1 of the first slice 6-1 described above. Specifically, as shown in FIG. 15, after the stage control unit 420 moves the specimen stage 20 to the imaging position P4-2 of the high-magnification image I4-2 of the second slice 6-2 (after step S200), the stage control unit 420 moves the imaging position from the imaging position P2-2 to the imaging position P4-2 of the high-magnification image I4-2 of the second slice 6-2 (step S202). The imaging unit 422 captures an alignment image I20-2 at the imaging position P4-2 (step S204).

[0093] The image processing unit 424 calculates the amount of positional deviation between the alignment image I20-2 and the medium-magnification image I2-2 (step S206), and calculates the difference D between the amount of movement V1 from the photographing position P2-2 to the photographing position P20-2 and the amount of positional deviation (dx2, dy2, dθ2) between the alignment image I20-2 and the medium-magnification image I2-2 (step S208). The movement amount V1 uses the same value as in the case of the first intercept 6-1. If the image processing unit 424 determines that the difference D is not smaller than the threshold value, If it is determined that the difference D is smaller (No in step S210), the stage control unit 420 corrects the photographing position P4-12 so that the difference D becomes smaller (step S212).

[0094] The processing unit 42 repeats steps S204 to S212 until the difference D becomes smaller than the threshold value. If the image processing unit 424 determines that the difference D is smaller than the threshold value (Yes in step S210), the photographing unit 422 photographs a high-magnification image I4-2 at the photographing position P4-2 (step S214).

[0095] The processing unit 42 repeats steps S200 to S218 until it is determined that M=N, and captures high-magnification images I4-1 to I4-N of each slice 6. When it is determined that M=N is satisfied (Yes in step S216), the imaging unit 422 ends the high-magnification image capturing process S18.

[0096] Effects In the electron microscope 100 according to the second embodiment, the control unit 40 performs the following processes: capturing a medium-magnification image I2-1 (an example of a first alignment image) at the shooting position P2-1 (an example of a first shooting position) of the medium-magnification image I2-1 of the first slice 6-1; capturing a alignment image I20-1 at the shooting position P4-1 (an example of a second shooting position) of the high-magnification image I4-1 of the slice 6-1; and determining the amount of positional deviation between the medium-magnification image I2-1 and the alignment image I20-1.

[0097] The control unit 40 also performs the following processes: determining whether the amount of movement V1 from the shooting position P2-1 to the shooting position P4-1 and the difference D in the amount of positional deviation between the medium-magnification image I2-1 and the alignment image I20-1 are smaller than a threshold; correcting the shooting position P4-1 so that the difference D becomes smaller if it is determined that the difference D is not smaller than the threshold; and capturing the alignment image I20-1 at the corrected shooting position P4-1. The control unit 40 also performs the following processes: obtaining the amount of positional deviation between the medium-magnification image I2-1 and the alignment image I20-1; determining whether the difference D between the amount of movement V1 and the deviation is smaller than a threshold; and correcting the shooting position P4-1 so that the difference D becomes smaller if it is determined that the difference D is not smaller than the threshold.

[0098] Therefore, in the electron microscope 100, the imaging position P4-1 for capturing the high-magnification image I4-1 can be accurately positioned relative to the imaging position P2-1 of the first slice 6-1, thereby reducing the positional deviation of the imaging position P4-1. Therefore, in the electron microscope 100, for example, there is no need to increase the imaging field of view for the high-magnification image in consideration of positional deviation, which makes it possible to shorten the imaging time, reduce the image size of the high-magnification image, and improve the accuracy of alignment during 3D reconstruction.

[0099] In the electron microscope 100, when the control unit 40 determines that the difference D is smaller than the threshold value, it performs a process of capturing a high-magnification image I4-1 at a magnification different from that of the alignment image I20-1 at the imaging position P4-1. Therefore, in the electron microscope 100, alignment between images can be performed using an alignment image at a magnification different from that of the high-magnification image, which is the target image. Therefore, the magnification of the alignment image can be set to a magnification suitable for aligning the images.

[0100] In the electron microscope 100, the control unit 40 sets the magnification of the alignment image I20-1 to be lower than the magnification of the high-magnification image I4-1 in the process of capturing the alignment image I20-1. Therefore, in the electron microscope 100, the possibility of losing sight of the field of view can be reduced compared to when alignment is performed using, for example, the high-magnification image I4-1.

[0101] 2.4. Variations Fig. 16 is a flowchart showing a modified example of the high-magnification image capturing process S18 in the electron microscope 100. Figs. 17 and 18 are diagrams for explaining each step of the high-magnification image capturing process. be.

[0102] In the second embodiment described above, there is one position for photographing a high-magnification image of each slice 6, but there may be multiple positions for photographing a high-magnification image of each slice 6. Below, a case will be described in which there are n positions for photographing a high-magnification image of each slice 6 (n is an integer of 2 or more).

[0103] First, the stage control unit 420 moves the specimen stage 20 to a position for capturing a medium-magnification image of the M-th slice 6 (step S200), and then captures the m-th high-magnification image I4 m m-th shooting position P4 at -1 m The photographing position is moved to -1 (step S202).

[0104] 17, the stage control unit 420 moves the specimen stage 20 to the photographing position P2-1 of the medium-magnification image I2-1 of the first slice 6-1 (step S200), and then moves the photographing position to the first photographing position P41-1 of the first high-magnification image I41-1 (step S202). The photographing unit 422 photographs the alignment image I201-1 (step S204), and the image processing unit 424 calculates the amount of positional deviation between the alignment image I201-1 and the medium-magnification image I2-1 (step S206), calculates the difference D1 between the amount of movement V1 from the photographing position P2-1 to the first photographing position P41-1 and the amount of positional deviation (step S208), and determines whether the difference D1 is smaller than a threshold (step S210).

[0105] If the image processing unit 424 determines that the difference D1 is not smaller than the threshold value (No in step S210), the stage control unit 420 corrects the first photographing position P41-1 (step S212) and returns to step S204. If the image processing unit 424 determines that the difference D1 is not smaller than the threshold value (No in step S210), the photographing unit 422 photographs the first high-magnification image I41-1 at the first photographing position P41-1 (step S214). In this way, the processing unit 42 performs each of steps S204 to S214 to photograph the first high-magnification image I41-1 at the first photographing position P41-1.

[0106] The photographing unit 422 determines whether photographing has been performed at all photographing positions of the first slice 6-1, i.e., whether m=n (step S215a), and if it determines that m=n is not true (No in step S215a), it sets m=m+1 (step S215b), returns to step S202, and as shown in Figure 18, the stage control unit 420 moves the photographing field of view to the second photographing position P42-1 of the second high-magnification image I42-1 (step S202). The photographing unit 422 photographs an alignment image I202-1 (step S204), and the image processing unit 424 calculates the amount of positional deviation between the alignment image I202-1 and the medium magnification image I2-1 (step S206), calculates the difference D2 between the amount of movement V2 from the photographing position P2-1 to the second photographing position P42-1 and the amount of positional deviation (step S208), and determines whether the difference D2 is smaller than a threshold value (step S210).

[0107] If the image processing unit 424 determines that the difference D2 is not smaller than the threshold value (No in step S210), the stage control unit 420 corrects the second photographing position P42-1 (step S212) and returns to step S204. If the image processing unit 424 determines that the difference D2 is not smaller than the threshold value (No in step S210), the photographing unit 422 photographs the second high-magnification image I42-1 at the second photographing position P42-1 (step S214). In this way, the processing unit 42 performs each of steps S204 to S214 to photograph the second high-magnification image I42-1 at the second photographing position P42-1.

[0108] The processing unit 42 repeats the steps S202 to S215b until it is determined that m=n, and outputs the first to n-th high-magnification images I41-1 to I41-4 of the first slice 6-1. n The imaging unit 422 images all of the imaging positions P41-1 to P4-1 of the first slice 6-1. n If it is determined that the image was taken at -1, that is, m=n (Yes in step S215a), it is determined whether M=N. If the imaging unit 422 determines that M is not equal to N (No in step S216), the processing unit 42 sets M to 2 and performs the same processing on the second slice 6-2 as on the first slice 6-1.

[0109] The processing unit 42 repeats steps S200 to S218 until it is determined that M=N, and captures n high-magnification images for each slice 6. When it is determined that M=N is satisfied (Yes in step S216), the imaging unit 422 ends the high-magnification image capturing process S18.

[0110] This modification can achieve the same effects as the electron microscope 100 according to the second embodiment described above. Furthermore, since multiple high-magnification images can be captured from one slice 6, multiple three-dimensional images can be generated from one serial slice 4.

[0111] 3. Third embodiment 3.1. Electron microscope Next, an electron microscope according to a third embodiment will be described. The configuration of the electron microscope according to the third embodiment is the same as that of the electron microscope shown in Fig. 1, and therefore a description thereof will be omitted.

[0112] Processing In the electron microscope 100 according to the third embodiment, high-magnification images are captured by combining the high-magnification image capturing process S18 in the first embodiment shown in Fig. 10 described above with the high-magnification image capturing process S18 in the second embodiment shown in Fig. 13 described above. Below, differences from the examples of the electron microscope 100 according to the first embodiment and the electron microscope 100 according to the second embodiment described above will be described, and similarities will not be described. Fig. 19 is a flowchart showing an example of the high-magnification image capturing process S18 in the electron microscope 100.

[0113] First, the stage control unit 420 moves the imaging position to imaging position P4-1 for a high-magnification image of the first slice 6-1 (step S300) and corrects the imaging position P4-1 (step S302), as shown in Fig. 14. The processing unit 42 corrects the imaging position P4-1 by performing steps S200 to S212 shown in Fig. 13.

[0114] Next, the photographing unit 422 photographs an alignment image I10-1 at the photographing position P4-1 (step S304), and photographs a high-magnification image I4-1 at the photographing position P4-1 (step S306), as shown in Fig. 11. The process S304 for photographing the alignment image I10-1 is similar to the process S102 shown in Fig. 10, and the process S306 for photographing the high-magnification image I4-1 is similar to the process S104 shown in Fig. 10, so detailed descriptions thereof will be omitted.

[0115] Next, the stage control unit 420 moves the sample stage 20 to an imaging position P4-2 where a high-magnification image I4-2 of the second slice 6-2 is captured (step S308), as shown in Fig. 15, and corrects the imaging position P4-2 (step S310). The processing unit 42 corrects the imaging position P4-2 by performing steps S200 to S212 shown in Fig. 13.

[0116] 12, the photographing unit 422 photographs an alignment image I10-2 at a photographing position P4-2 (step S312), and the image processing unit 424 calculates the amount of misalignment between the alignment images I10-1 and I10-2 (step S314). The image processing unit 424 determines whether the calculated amount of misalignment is smaller than a threshold (step S316), and if it is determined that the amount of misalignment is not smaller than the threshold (No in step S316), the stage control unit 420 corrects the photographing position P4-2 (step S318). The processing unit 42 repeats steps S312 to S318 until it is determined that the amount of misalignment between the alignment images I10-1 and I10-2 is smaller than the threshold.

[0117] If the image processing unit 424 determines that the amount of positional deviation is smaller than the threshold value (Yes in step S316), the photographing unit 422 photographs a high-magnification image I4-2 at the photographing position P4-2 (step S320). This allows the high-magnification image I4-2 to be photographed in the second slice 6-2. The processes in steps S312 to S320 described above are similar to the processes in steps S108 to S116 shown in FIG. 10, and therefore detailed descriptions thereof will be omitted.

[0118] The photographing unit 422 determines whether high-magnification images have been photographed for all slices 6, i.e., whether M=N (step S322), and if it is determined that M=N is not true (No in step S322), it sets M=M+1 and performs steps S308 to S320 to photograph a high-magnification image of the next slice 6. The processing unit 42 repeats steps S308 to S320 until it is determined that high-magnification images have been photographed for all slices 6, photographing high-magnification images I4-1 to I4-N of each slice 6.

[0119] When the photographing unit 422 determines that high-magnification images have been photographed for all slices 6, that is, that M=N is satisfied (Yes in step S322), the high-magnification image photographing process S18 ends.

[0120] Effects The electron microscope 100 according to the third embodiment can achieve the same effects as the electron microscope 100 according to the first embodiment and the electron microscope 100 according to the second embodiment described above.

[0121] 4. Fourth embodiment 4.1. Electron microscope Next, an electron microscope according to a fourth embodiment will be described. The configuration of the electron microscope according to the fourth embodiment is the same as that of the electron microscope shown in Fig. 1 described above, and therefore a description thereof will be omitted.

[0122] Processing In the first to third embodiments described above, the case where the imaging position is corrected between different sections 6 has been described. However, even when imaging the same section at the same imaging position, the field of view may shift due to a change in imaging conditions. For example, imaging conditions that cause the field of view to shift include the acceleration voltage, the distance between the objective lens and the sample (working distance), the bias voltage applied to the sample stage 20, and switching between IN and OUT of the retractable detector. Therefore, in the electron microscope 100 according to the fourth embodiment, the imaging position is corrected after switching the observation conditions, and high-magnification images of the same field of view are captured under different imaging conditions.

[0123] Fig. 20 is a flowchart showing an example of high-magnification image capturing processing in the electron microscope 100. Fig. 21 is a diagram for explaining the high-magnification image capturing processing. Below, a case will be described in which, after a high-magnification image is captured under normal capturing conditions, a high-magnification image is captured at the same capturing position with a bias voltage applied to the sample stage 20.

[0124] First, the imaging unit 422 captures a high-magnification image I100 at the imaging position P0 of the first slice 6-1 (step S400). The magnification of the high-magnification image I100 is, for example, 50,000 times.

[0125] Next, the photographing unit 422 photographs a first alignment image I200 at the photographing position P0 (step S402). The magnification of the first alignment image I200 is lower than that of the high-magnification image I100. The magnification of the first alignment image I200 is, for example, 10,000 times. The photographing conditions of the first alignment image I200 are the same as those of the high-magnification image I100, except for the magnification. This is the same as the shooting conditions for 100.

[0126] Next, the photographing unit 422 photographs the second alignment image I202 at the photographing position P0 (step S404). The magnification of the second alignment image I202 is lower than that of the first alignment image I200. The magnification of the second alignment image I202 is, for example, 1000 times. The photographing conditions for the second alignment image I202 are the same as those for the first alignment image I200, except for the magnification.

[0127] Next, the imaging unit 422 changes the imaging conditions (step S406). Here, the imaging conditions are set to a BD (Beam Deceleration) mode in which a bias voltage is applied to the sample stage 20. In the BD mode, a bias voltage is applied to the sample stage 20, thereby decelerating the incident electrons just before they reach the sample. This makes it possible to improve the spatial resolution and the signal-to-noise ratio.

[0128] Next, the photographing unit 422 photographs the third alignment image I204 at the photographing position P0 (step S408). Here, the third alignment image I204 is photographed without moving the sample stage 20 after photographing the second alignment image I202. The photographing conditions for the third alignment image I204 are the same as those for the second alignment image I202, except that the photographing conditions are the BD mode.

[0129] Here, the position of the sample stage 20 when the second alignment image I202 is captured is the same as the position of the sample stage 20 when the third alignment image I204 is captured, but a positional shift occurs between the second alignment image I202 and the third alignment image I204 due to switching to BD mode.

[0130] Therefore, the image processing unit 424 calculates the amount of misalignment between the second alignment image I202 and the third alignment image I204 (step S410), and determines whether the amount of misalignment is smaller than a first threshold (step S412). If the image processing unit 424 determines that the amount of misalignment is not smaller than the first threshold (No in step S412), the stage control unit 420 corrects the shooting position P0 so that the amount of misalignment becomes smaller (step S414).

[0131] After the stage control unit 420 corrects the photographing position P0 (after step S414), the process returns to step S408, and the photographing unit 422 photographs the third alignment image I204 at the corrected photographing position P0. In this way, the processing unit 42 repeats steps S408 to S414 until it is determined that the amount of positional deviation between the second alignment image I202 and the third alignment image I204 is smaller than the first threshold. Note that steps S408 to S414 are similar to steps S108 to S114 shown in FIG. 10, and therefore detailed description thereof will be omitted.

[0132] If the image processing unit 424 determines that the amount of misalignment between the second alignment image I202 and the third alignment image I204 is smaller than the first threshold (Yes in step S412), a fourth alignment image I206 is captured (step S416). The fourth alignment image I206 is captured at the capturing position P0 corrected by steps S408 to S414 described above. The capturing conditions for the fourth alignment image I206 are the same as those for the first alignment image I200, except that it is in BD mode.

[0133] The image processing unit 424 calculates the amount of misalignment between the first alignment image I200 and the fourth alignment image I206 (step S418), and determines whether the amount of misalignment is smaller than a second threshold value (step S420). The second threshold value may be set to a value different from the first threshold value, or may be set to the same value as the first threshold value. If the image processing unit 424 determines that the amount of misalignment is not smaller than the second threshold value (No in step S420), the stage control unit 420 The photographing position P0 is corrected so as to reduce the amount of misalignment (step S422). After the stage control unit 420 corrects the photographing position P0 (after step S422), the process returns to step S416, and the photographing unit 422 photographs the fourth alignment image I206 at the corrected photographing position P0. Since the processes of steps S416 to S422 are the same as the processes of steps S108 to S114 shown in Fig. 10, detailed description thereof will be omitted.

[0134] If the image processing unit 424 determines that the amount of positional deviation between the first alignment image I200 and the fourth alignment image I206 is smaller than the second threshold value (Yes in step S420), a high-magnification image I102 in BD mode is captured (step S424). The high-magnification image I102 in BD mode is captured at the imaging position P0 corrected by the above-mentioned steps S408 to S414 and the above-mentioned steps S416 to S422. The imaging conditions for the high-magnification image I102 in BD mode are the same as those for the high-magnification image I100, except that it is in BD mode.

[0135] The image processing unit 424 calculates the amount of positional deviation between the high-magnification image I100 and the high-magnification image I102 (step S426) and determines whether the amount of positional deviation is smaller than a third threshold (step S428). The third threshold may be set to a value different from or equal to the first and second thresholds. If the image processing unit 424 determines that the amount of positional deviation is not smaller than the third threshold (No in step S428), the stage control unit 420 corrects the photographing position P0 so as to reduce the amount of positional deviation (step S430). After the stage control unit 420 corrects the photographing position P0 (after step S430), the process returns to step S424, and the photographing unit 422 photographs the high-magnification image I102 at the corrected photographing position P0. The processes in steps S424 to S430 are similar to the processes in steps S108 to S114 shown in FIG. 10, and therefore detailed descriptions thereof will be omitted.

[0136] If the image processing unit 424 determines that the amount of positional deviation between the high-magnification image I100 and the high-magnification image I102 is smaller than the third threshold value (Yes in step S428), the process ends.

[0137] Effects In the electron microscope 100 according to the fourth embodiment, the control unit 40 performs a process of capturing a high-magnification image at a first magnification (an example of a first target image), a process of capturing a first alignment image I200 at a second magnification lower than the first magnification, a process of capturing a second alignment image I202 at a third magnification lower than the second magnification, and a process of switching the imaging conditions.

[0138] The control unit 40 also performs a process S204 of capturing a third alignment image I204 at a third magnification, a process of calculating a first shift amount between the second alignment image I202 and the third alignment image I204 and determining whether the first shift amount is smaller than a first threshold value, and a process of correcting the capturing position P0 so that the first shift amount becomes smaller if it is determined that the first shift amount is not smaller than the first threshold value.

[0139] In addition, if the control unit 40 determines that the first shift amount is smaller than the first threshold, it performs the following processes: a process of capturing a fourth alignment image I206 at a second magnification; a process of calculating a second shift amount between the first alignment image I200 and the fourth alignment image I206 and determining whether the second shift amount is smaller than the second threshold; a process of correcting the shooting position P0 so that the second shift amount becomes smaller if it determines that the second shift amount is not smaller than the second threshold; and a process of capturing a high-magnification image I102 at a first magnification (an example of a second target image) if it determines that the second shift amount is smaller than the second threshold.

[0140] Therefore, in the electron microscope 100, after correcting the photographing position P0 using the second alignment image I202 and the third alignment image I204, the first alignment image I200 and the fourth alignment image I203 are used. Since the photographing position is corrected using the alignment image I206, the photographing position P0 can be accurately corrected, thereby reducing the positional deviation between the high-magnification image I100 photographed before switching the photographing conditions and the high-magnification image I102 photographed after switching the photographing conditions.

[0141] Furthermore, the electron microscope 100 corrects the photographing position using a plurality of sets of alignment images (a set of the second alignment image I202 and the third alignment image I204, and a set of the first alignment image I200 and the fourth alignment image I206). This reduces the possibility of losing track of the field of view when aligning two images, and improves the accuracy of alignment. For example, when the magnification of the alignment image is high, the accuracy of alignment can be improved, but the narrow field of view can cause the field of view to be lost, making alignment impossible. Furthermore, when the magnification of the alignment image is low, the wide field of view can reduce the possibility of losing track of the field of view, but the accuracy of alignment is low. In contrast, the electron microscope 100 corrects the photographing position between the slices 6 using a plurality of sets of alignment images with different magnifications, thereby reducing the possibility of losing track of the field of view and improving the accuracy of alignment.

[0142] 4.4. Variations In the above-described fourth embodiment, the high-magnification image capturing process shown in FIG. 20 is performed on one of the sections 6 constituting the serial section 4 to capture high-magnification images of the same field of view under different capturing conditions. However, the electron microscope 100 may also perform the high-magnification image capturing process shown in FIG. 20 on a sample other than a serial section to capture high-magnification images of the same field of view under different capturing conditions.

[0143] In addition, in the above-mentioned fourth embodiment, in order to capture a high magnification image of 50,000x, the shooting position was corrected using a set of 10,000x alignment images and a set of 1,000x alignment images, but the shooting position may also be corrected using a set of three or more alignment images with different magnifications.

[0144] 5. Fifth embodiment 5.1. Electron microscope Next, an electron microscope according to a fifth embodiment will be described. The configuration of the electron microscope according to the fifth embodiment is the same as that of the electron microscope shown in Fig. 1 described above, and therefore a description thereof will be omitted.

[0145] Processing In the high-magnification image capturing process in the first embodiment described above, the capturing positions between sections are corrected using one set of alignment images, but in the electron microscope 100 according to the fifth embodiment, the capturing positions between sections are corrected using a plurality of sets of alignment images, as in the fourth embodiment described above. Below, differences from the examples of the electron microscope 100 according to the first to fourth embodiments described above will be described, and similarities will not be described.

[0146] Fig. 22 is a flowchart showing an example of the high-magnification image capturing process S18 in the electron microscope 100. Fig. 23 and Fig. 24 are diagrams for explaining the high-magnification image capturing process.

[0147] First, the stage control unit 420 moves the sample stage 20 to an imaging position where a high-magnification image of the Mth slice is captured (step S500). Here, M=1, and the imaging position is moved to imaging position P4-1 where a high-magnification image I4-1 of the first slice 6-1 is captured.

[0148] Next, as shown in FIG. 23, the photographing unit 422 photographs a first alignment image I200 at a photographing position P4-1 (step S502), and photographs a second alignment image I202 at a photographing position P4-1 (step S504). The photographing conditions for the second alignment image I202 are the same as those for the first alignment image I200, except that the magnification is lower than that of the first alignment image I200. The photographing conditions are the same as those for the first alignment image I200. The photographing unit 422 photographs a high-magnification image I4-1 at the photographing position P4-1 (step S506).

[0149] Next, the stage control unit 420 moves the sample stage 20 to an imaging position P4-2 where a high-magnification image I4-2 of the second slice 6-2 is captured (step S508).

[0150] Next, the photographing unit 422 photographs the third alignment image I204 at the photographing position P4-2 as shown in FIG. 24 (step S510). The image processing unit 424 calculates the amount of misalignment between the second alignment image I202 and the third alignment image I204 (step S512) and determines whether the amount of misalignment is smaller than a first threshold (step S514). If the image processing unit 424 determines that the amount of misalignment is not smaller than the first threshold (No in step S514), the stage control unit 420 corrects the photographing position P4-2 so as to reduce the amount of misalignment (step S516). After the stage control unit 420 corrects the photographing position P4-2 (after step S516), the process returns to step S510, and the photographing unit 422 photographs the third alignment image I204 at the corrected photographing position P4-2 (step S510). In this way, the processing unit 42 repeats steps S510 to S516 until it is determined that the amount of misalignment between the second alignment image I202 and the third alignment image I204 is smaller than the first threshold value.

[0151] If the image processing unit 424 determines that the amount of misalignment between the second alignment image I202 and the third alignment image I204 is smaller than the first threshold (Yes in step S514), a fourth alignment image I206 is captured (step S518). The fourth alignment image I206 is captured at the capturing position P4-2 corrected by steps S510 to S516 described above.

[0152] The image processing unit 424 calculates the amount of misalignment between the first alignment image I200 and the fourth alignment image I206 (step S520) and determines whether the amount of misalignment is smaller than the second threshold (step S522). If the image processing unit 424 determines that the amount of misalignment is not smaller than the second threshold (No in step S522), the stage control unit 420 corrects the photographing position P4-2 so that the amount of misalignment is smaller (step S524). After the stage control unit 420 corrects the photographing position P4-2 (after step S524), the process returns to step S518, and the photographing unit 422 photographs the fourth alignment image I206 at the corrected photographing position P4-2 (step S518). In this way, the processing unit 42 repeats steps S518 to S524 until it is determined that the amount of misalignment between the first alignment image I200 and the fourth alignment image I206 is smaller than the second threshold.

[0153] The processes of steps S510 to S524 are similar to the processes of steps S408 to S422 shown in FIG. 20, and therefore detailed description thereof will be omitted.

[0154] If the image processing unit 424 determines that the amount of positional deviation between the first alignment image I200 and the fourth alignment image I206 is smaller than the second threshold (Yes in step S522), a high-magnification image I4-2 is captured at the corrected shooting position P4-2 (step S526).

[0155] If the photographing unit 422 determines that high-magnification images have not been photographed for all slices 6, that is, that M=N is not satisfied (No in step S528), it sets M=M+1 (step S530) and returns to step S508 to perform process S508, in which the specimen stage 20 is moved to the photographing position for the third slice 6. The photographing unit 422 repeats processes S508 to S524 until it determines that high-magnification images have been photographed for all slices 6, that is, that M=N is satisfied.

[0156] When the photographing unit 422 determines that high-magnification images have been photographed for all slices 6, that is, that M=N is satisfied (Yes in step S526), ​​the high-magnification image photographing process S18 ends.

[0157] Effects In the electron microscope 100 according to the fifth embodiment, a set of multiple alignment images is used to correct the photographing positions between the slices 6, thereby reducing the positional deviation between the high-magnification images photographed for each slice 6. Furthermore, the same effects as those of the electron microscope 100 according to the fourth embodiment can be achieved.

[0158] 6. Variations The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the present invention.

[0159] For example, in the electron microscope 100 according to the first to fifth embodiments, the display unit 46 may display a GUI screen for inputting the alignment accuracy between high-magnification images. The imaging unit 422 sets the magnification and threshold of the alignment image based on the input alignment accuracy. This allows alignment to be performed using an alignment image with a magnification that corresponds to the alignment accuracy required by the user.

[0160] The same applies to the second to fifth embodiments described above, and the magnification and threshold value of the image for alignment may be set according to the accuracy of alignment input by the user.

[0161] The above-described embodiment and modifications are merely examples, and the present invention is not limited to these. For example, the embodiments and modifications can be combined as appropriate.

[0162] The present invention is not limited to the above-described embodiments, and various modifications are possible. For example, the present invention includes configurations that are substantially identical to the configurations described in the embodiments. A substantially identical configuration means, for example, a configuration with the same function, method, and result, or a configuration with the same purpose and effect. The present invention also includes configurations in which non-essential parts of the configurations described in the embodiments are replaced. The present invention also includes configurations that achieve the same effects or purposes as the configurations described in the embodiments. The present invention also includes configurations in which publicly known technology is added to the configurations described in the embodiments. [Explanation of symbols]

[0163] 10...electron optical system, 20...sample stage, 30...detector, 40...controller, 42...processor, 44...operator, 46...display, 48...storage unit, 100...electron microscope, 420...stage controller, 422...photographer, 424...image processor

Claims

1. A scanning electron microscope for photographing and three-dimensionally reconstructing each of a series of consecutive sections, each of which is a series of consecutive sections cut out from a sample, comprising: a sample stage for positioning the serial sections; an electron optical system that scans each of the slices with an electron beam; a detector for detecting electrons emitted by scanning each of the slices with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit a process of moving the sample stage to a first imaging position of a first slice; A process of capturing a first alignment image at the first imaging position; a process of moving the sample stage to a second imaging position for a second slice; A process of capturing a second alignment image at the second imaging position; a process of calculating a first amount of deviation between the first alignment image and the second alignment image, and determining whether the first amount of deviation is smaller than a first threshold value; a process of correcting the second photographing position so as to reduce the first deviation amount when it is determined that the first deviation amount is not smaller than the first threshold value; A process of capturing a third alignment image at the corrected second imaging position; a process of calculating a second amount of deviation between the first alignment image and the third alignment image, and determining whether the second amount of deviation is smaller than the first threshold value; Scanning electron microscope.

2. In claim 1, The control unit a process of capturing a first target image at the first photographing position, the first target image having a magnification different from that of the first alignment image; a process of capturing a second target image at the second photographing position at a magnification different from that of the second alignment image when it is determined that the first deviation amount is smaller than the first threshold value; Scanning electron microscope.

3. In claim 2, The control unit, in the process of capturing the first alignment image, A scanning electron microscope, wherein the magnification of the first alignment image is set lower than the magnification of the first target image.

4. A scanning electron microscope for photographing and three-dimensionally reconstructing each of a series of consecutive sections, each of which is a series of consecutive sections cut out from a sample, comprising: a sample stage for positioning the serial sections; an electron optical system that scans the slice with an electron beam; a detector for detecting electrons emitted by scanning the slice with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit A process of capturing a first alignment image at a first capturing position of the first slice; capturing a second alignment image at a second imaging position different from the first imaging position of the first slice; A process of calculating a first deviation amount between the first alignment image and the second alignment image; A first difference between the amount of movement from the first photographing position to the second photographing position and the first amount of deviation is a first threshold. a process of determining whether the value is smaller than the a process of correcting the second photographing position so as to reduce the first difference when it is determined that the first difference is not smaller than the first threshold value; A process of capturing a third alignment image at the corrected second imaging position; A process of calculating a second deviation amount between the first alignment image and the third alignment image; a process of determining whether a second difference between the movement amount and the second deviation amount is smaller than the first threshold value; a process of correcting the second imaging position so as to reduce the second difference when it is determined that the second difference is not smaller than the first threshold value; Scanning electron microscope.

5. In claim 4, When the control unit determines that the first difference is smaller than the first threshold, the control unit performs a process of capturing a target image at the second shooting position at a magnification different from that of the second alignment image.

6. In claim 5, In the process of capturing the second alignment image, the control unit A scanning electron microscope, wherein the magnification of the second alignment image is set lower than the magnification of the target image.

7. a sample stage for positioning the sample; an electron optical system that scans the sample with an electron beam; a detector for detecting electrons emitted by scanning the sample with an electron beam; a control unit that controls the sample stage and the electron optical system; Including, The control unit A process of capturing a first target image at a first imaging position and a first magnification; a process of capturing a first alignment image at a second magnification lower than the first magnification; capturing a second alignment image at a third magnification that is lower than the second magnification; Processing to switch shooting conditions, a process of capturing a third alignment image at the third magnification; a process of calculating a first deviation amount between the second alignment image and the third alignment image, and determining whether the first deviation amount is smaller than a first threshold value; a process of correcting the first imaging position so as to reduce the first deviation amount when it is determined that the first deviation amount is not smaller than the first threshold value; capturing a fourth alignment image at the second magnification when it is determined that the first deviation amount is smaller than the first threshold value; a process of calculating a second deviation amount between the first alignment image and the fourth alignment image, and determining whether the second deviation amount is smaller than a second threshold value; a process of correcting the first photographing position so as to reduce the second deviation amount when it is determined that the second deviation amount is not smaller than the second threshold value; capturing a second target image at the first magnification when it is determined that the second deviation amount is smaller than the second threshold value; Scanning electron microscope.

8. In claim 7, The control unit performs processing to determine the amount of positional deviation between the first target image and the second target image.

Images