WO2018189850A1

WO2018189850A1 - Electron microscope

Info

Publication number: WO2018189850A1
Application number: PCT/JP2017/015072
Authority: WO
Inventors: 長沖　功; 圭司田村; 孝史藤井; 小林　隆幸; 甲子男影山; 勉和田; 大海三瀬; 暁哉広田; 稲田　宏; 拓上ノ内
Original assignee: 株式会社日立ハイテクノロジーズ
Priority date: 2017-04-13
Filing date: 2017-04-13
Publication date: 2018-10-18

Abstract

Conventionally, in order to observe the entirety of a sample, the objective lens current is lowered to 1/6 of the current value used normally, reducing the magnetic field in the vicinity of the sample and achieving 50-fold magnification. Because of this, when a normally used observation magnification is set, the rising current is a factor in the occurrence of sample drift. In order to solve the problem, an electron microscope is provided, comprising an electron source irradiating an electron beam onto a sample, and a detector detecting electrons arising from the sample due to the electron beam irradiation, characterized in that, in the electron microscope, an upper objective lens coil and a lower objective lens coil are provided above and below the sample, the polarities of the upper objective lens coil and the lower objective lens coil being inverted with respect to one another.

Description

electronic microscope

The present invention relates to an electron microscope.

There are various types of microscopes, and they are used in a wide range of applications such as nanotechnology, materials, and medicine / biology. In the medical / biological field, an electron microscope is used to elucidate the fine structure of cells and tissues. On the other hand, in order to observe the localization and behavior of proteins at the molecular level, a fluorescence microscope, which is a type of optical microscope, is used. Progressing.
In recent years, light-electron correlation microscopy (hereinafter referred to as CLEM), in which observation is performed using different types of microscopes, such as electron microscopes and fluorescence microscopes, has been actively used. CLEM analyzes the location specific to a molecule with nano-level resolution by observing the location observed with a fluorescence microscope with an electron microscope, comparing the observation images obtained from the two methods, and correlating them. It is expected that.

In order to perform this type of observation, not only optical microscope images but also electron microscope LOWMAG (low-magnification observation), a PC-controlled pneumatic drive objective aperture, and image processing technology that superimposes images are provided. Yes.

Patent Document 1 describes an apparatus provided with an optical microscope in a transmission electron microscope.

Japanese Patent Laying-Open No. 2015-141899

In sample observation using an electron microscope, CLEM that improves the inspection efficiency by correlating an optical microscope image and an electron microscope image is important in the field of medical biology. However, it is not easy to observe the same part with microscopes having different magnification areas and observation items. Electron microscopes for medical organisms are compatible with CLEM, and frequently switch between 50x ultra-low magnification images close to light microscopic images and high-definition observations. Conventionally, when the entire sample is viewed, the objective lens current is reduced to about 1/6 of the current value that is normally used, and the magnetic field is reduced in the vicinity of the sample to achieve 50 times. For this reason, when the observation magnification used normally is used, the current increases, causing a sample drift.

In the case of a transmission electron microscope as described in Patent Document 1, it is necessary to insert or remove the objective aperture on the optical axis when switching from high-definition observation to ultra-low magnification observation, and a mechanism capable of PC control is necessary. Yes.

The object of the present invention is to provide a constant energy by inverting the polarity of the coil on one side in an objective lens in which coils of the same number of turns are arranged on the upper and lower sides of the sample and pole piece and an auxiliary lens is incorporated in the magnetic path. The objective is to provide an electron microscope that can reduce the sample drift by reducing the magnetic field in the vicinity of the sample and realizing an extremely low magnification of 50 times. According to the present embodiment, the degree of coincidence can be calculated by performing correlation calculation using an overlay technique for matching the optical microscope and the electron microscope image.

In order to solve the above problems, an embodiment of the present invention is an electron microscope including an electron source for irradiating a sample with an electron beam and a detector for detecting electrons generated from the sample by the electron beam irradiation. Provided is an electron microscope comprising an upper objective lens coil and a lower objective lens coil, wherein the polarities of the upper objective lens coil and the lower objective lens coil are reversed.

According to the present invention, observation efficiency is improved because image observation with less drift can be performed by switching between a 50 × ultra-low magnification image close to a light microscope image and high-definition observation.

1 is a schematic configuration diagram of a scanning fluoroscopic electron microscope according to an embodiment of the present invention. The figure which displayed the ultra-low magnification electron microscope image based on embodiment of this invention. On-axis magnetic field and magnetic saturation diagram at objective current value at extremely low magnification according to an embodiment of the present invention The on-axis magnetic field and magnetic saturation diagram when the object polarity is switched at the time of extremely low magnification according to the embodiment of the present invention. The flowchart figure of the seamless function which concerns on embodiment of this invention. The flowchart figure of the seamless function and objective aperture control which concern on embodiment of this invention. GUI image of seamless function according to an embodiment of the present invention The GUI image of the seamless function and objective aperture control which concern on embodiment of this invention. An example of the CLEM display screen which concerns on embodiment of this invention. The flowchart figure of the image superposition display which concerns on embodiment of this invention. An example of the image superimposition display screen which concerns on embodiment of this invention. An example of the CLEM display screen which concerns on embodiment of this invention. An example of displaying an optical microscope image according to an embodiment of the present invention on an electron microscope control GUI

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following, a transmission electron microscope (TEM) will be described as an example, but the present invention is an electron microscope including a scanning electron microscope (SEM), a scanning transmission electron microscope (STEM), and a scanning transmission electron microscope. It can be applied not only to a charged particle beam apparatus.

FIG. 1 is a schematic configuration diagram of a transmission electron microscope according to an embodiment of the present invention. The electron microscope apparatus shown in this figure includes an electron gun 1, first and second

irradiation lens coils

2 and 3, first and second deflection coils (scanning coils) 4 and 5, and an objective lens coil 6. , First and second electromagnetic sample

image moving coils

7, 8, first and second intermediate lens coils 9, 10, first and second projection lens coils 11, 12, and excitation power supply 13 23, digital-analog converters (DACs) 24 to 34, a microprocessor (MPU) 35, a hard disk drive (HDD) 36, an arithmetic unit (ALU) 37, a monitor controller (CRT controller) 38, A monitor (CRT) 39, interfaces (I / F) 40 and 41, a magnification switching rotary encoder (RE) 42, an input rotary encoder (RE) 43, and a key And over de 44, a mouse 57, and a RAM 45, a ROM 46, an interface 48 image capture. A sample holder 53 for holding the sample 70 (see FIG. 3) is disposed on the optical axis. The

objective lens coils

6 and 7 shown in the figure are strong excitation lenses (see FIGS. 3 and 4), and lenses are formed on the upper and lower sides of the sample.

FIG. 2 is a diagram showing a GUI displaying an extremely low magnification image related to the present invention in the electron microscope apparatus according to the embodiment of the present invention. In this figure, a typical electron microscope sample is placed on a 3 mm mesh and placed on a sample holder for observation. For this reason, the maximum field of view is 2 mm because the surrounding 0.5 mm is pressed by the sample holder. The electron microscope section according to the present embodiment is configured so that the electron gun 1, the converging electron lens formed by the

irradiation lens coils

2 and 3, and the electron beam generated by the electron gun 1 are placed on the sample 70 (see FIG. 3). A transmission electron microscope image is formed by the

deflection coils

4 and 5 for adjusting the brightness, the sample holder 53 holding the sample 70, the objective lens for imaging the sample, and the objective auxiliary lens 8. The formed transmission electron microscope image is magnified by the intermediate lenses 9 and 10 and the imaging lenses 11 and 12, and is displayed on the fluorescent screen 49 or the TV camera 56. A part of the hardware shown in FIG. 1 such as the sensor 35, HDD 36, monitor controller 38, RAM 45, ROM 46, and image capture interface 48, a recording control device 95 for recording a microscopic image displayed on the monitor 39, etc. Is installed.

FIG. 3 is a detailed view of the objective lens, in which the objective lens coils 6 and 7 are arranged vertically symmetrically, and the auxiliary lens 8 is arranged in the magnetic path. When an extremely low magnification image (LOWMAG) is formed, it is usually used only by irradiating the sample with an electron beam after flowing a small current through the

objective lens coils

6 and 7 to reduce the axial magnetic field. The value of current that flows through the objective lens when forming a very low magnification image (LOWMAG) is about 1/10 or less of the value of current that flows through the objective lens when forming a high-definition image (ZOOM). Therefore, when moving from a very low magnification image (LOWMAG) to a high definition image (ZOOM), a large difference occurs in the energy of the objective lens coil. Due to this energy difference, a temperature change occurs and sample drift occurs, so that the field of view may move when switching from very low magnification observation to high definition observation.

FIG. 4 shows an objective lens according to an embodiment of the present invention, in which objective lens coils 6 and 7 are vertically symmetrically arranged with an upper objective lens coil 6 and a lower objective lens coil 7, and an auxiliary lens 8 is provided in the magnetic path. It is arranged. In the present invention, an on-axis magnetic field when the polarity of the objective lens coil 7 is reversed with respect to the objective lens coil 6 is shown. Since a magnetic field for forming a very low magnification image (LOWMAG) can be formed, an extremely low magnification image can be obtained with a small amount of current flowing through the

objective lens coils

6 and 7 from which a high definition image (ZOOM) can be obtained. Since there is no difference, the temperature remains constant and sample drift does not occur. The polarity of the upper objective lens coil and the lower objective lens coil are reversed, and the field of view is searched with the magnetic field cancelled, so the field of view does not move even when moving from extremely low magnification observation to high definition observation. The sample can be easily observed.

The temperature of the coil does not change because the magnetic field around the sample is reduced to such an extent that the electron beam applied to the sample by the auxiliary lens can be collimated and the current value of the lens coil is constant at extremely low magnification and high-definition observation. . The polarity of the upper objective lens coil and the lower objective lens coil may be reversed and controlled by a control unit or the like so as to cancel the magnetic field.

FIG. 5 is a flowchart according to the embodiment of the present invention, and uses an extremely low magnification image (LOWMAG) in which the polarity of the objective lens coil in FIG. 4 is reversed. When seamlessly ON, it is possible to shift from a high-definition image (ZOOM) to a very low-magnification image (LOWMAG) while keeping the current of the objective lens coil constant. Therefore, it is possible to observe the entire field of view at the minimum magnification of 50. When seamless is turned off, it stops at the minimum magnification of ZOOM mode (high definition mode), and the entire field of view can be obtained in the same way when switching to LOWMAG (very low magnification image). Note that the control unit or the like may perform control to automatically switch from the high-definition observation mode to the ultra-low magnification observation mode.

FIG. 6 is a flowchart according to the embodiment of the present invention, and uses an extremely low magnification image (LOWMAG) in a state where the polarity of the objective lens coil in FIG. 4 is reversed. When both seamless ON and objective aperture automatic control are used together, the objective lens coil current is kept constant from ZOOM to LOWMAG, and the objective aperture is automatically output to enable observation of the entire field of view with a minimum magnification of 50. . When seamless is turned off, it stops at the lowest magnification in ZOOM mode, and the entire field of view can be obtained in the same way as switching to LOW MAG. In this case, it is necessary to manually turn the objective aperture out of the optical axis.

In addition, the control unit controls the objective aperture so that it can control IN / OUT from the optical axis, and the objective aperture is adjusted to automatically switch from the high-definition observation mode to the ultra-low magnification observation mode. It may be configured to perform IN / OUT control.

FIG. 7 is a control GUI example according to the embodiment of the present invention, and the operation of FIG. 5 can be performed by checking the Seamless Zoom check box.

FIG. 8 is an example of a control GUI according to the embodiment of the present invention, and the operation of FIG. 6 can be performed by checking the checkbox of Seamless チェック Zoom and IN / OUT of OBJ.ap.

Fig. 9 shows an example of a pneumatic drive objective diaphragm mechanism. It is possible to in / out the objective diaphragm from the optical axis with air.

FIG. 10 shows an example in which an optical microscope image is displayed on the electron microscope control GUI in the control GUI example according to the embodiment of the present invention. Since an optical microscope usually has color information, it can be displayed in color on the GUI.

FIG. 11 is a flowchart according to the embodiment of the present invention, and obtains magnification data from an optical microscope and performs image composition processing. First, image enlargement processing is performed, and then magnification data is acquired. After that, the image composition processing loop is executed to acquire the image data in the “TemMap” folder. Next, the size of the acquired image data is calculated and the image size is confirmed. At this time, if the image size is 10 pixels or more and less than 512 pixels, the image position is calculated and combined with the Stageimage area. After that, an image composition processing loop is performed, the Stageimage area is redrawn, and the enlargement process ends. At this time, if the overlay technique for matching the optical microscope image and the electron microscope image, which will be described below, is used, it is possible to automatically calculate the amount of movement of X and Y. You can also.

Next, a superposition technique for matching an optical microscope image and an electron microscope image by calculating a specific movement amount and calculating a matching degree according to the embodiment of the present invention will be described.

(1) Means for calculating movement amount First, the operation of the transmission electron microscope having the above-described configuration will be described using an example of image correlation. An image obtained by cutting out a part of the transmission image 1 is recorded as a transmission image 3 as a registered image f1 (m, n) in the storage device with the number of pixels of M × N. Next, the image captured after the recording mode is recorded as f2 (m, n) as a reference image in the storage device with the transmission image 2 having the number of pixels of M × N.

However, both are natural images, and m = 0, 1, 2,... M-1 and n = 0, 1, 2,.

Discrete Fourier images F1 (m, n) and F2 (m, n) of f1 (m, n) and f2 (m, n) are defined by (Equation 1) and (Equation 2), respectively.
(Equation 1) F1 (u, v) = A (u, v) ejθ (u, v)
(Expression 2) F2 (u, v) = B (u, v) ejφ (u, v)
However, u = 0,1,2 ... M-1, V = 0,1,2, ... N-1
A (u, v) and B (u, v) are amplitude spectra, and θ (u, v) and φ (u, v) are phase spectra.

In phase correlation, if there is parallel movement of an image between two images, the position of the correlation peak is shifted by the amount of movement.

The following explains how to derive the movement amount.

First, assuming that the original image f2 (m, n) has moved by r ′ in the x direction, f4 (m, n) = f2 (m + r ′, n).

(Formula 2) is transformed into (Formula 3).
(Expression 3) F4 (u, v) = ΣΣ f2 (m + r ′, n) e−j2π (mu / M + nv / N)
= B (u, v) ej (φ + 2πr'u / M)
By setting the amplitude spectrum B (u, v) as a constant, a phase image independent of the contrast of the image is obtained. The phase image F′4 (u, v) of f4 is expressed by equation (4).
(Equation 4) F4 ′ (u, v) = ej (φ + 2πr′u / M)
By multiplying the phase image F′1 (u, v) by the complex function of F′2 (u, v), the synthesized image H14 (u, v) becomes (Expression 5).
(Expression 5) H14 (u, v) = F'1 (u, v) (F'2 (u, v)) *
= Ej (θ-φ-2πru / M)
The correlation intensity image G14 (r, s) is expressed by (Equation 6) by performing inverse Fourier transform on the composite image H14 (u, v).
(Expression 6) G14 (r, s) = ΣΣ (H14 (u, v)) ej2π (ur / M + us / N)
= ΣΣ (ej (θ-φ-2πr'u / M)) ej2π (ur / M + us / N)
= G12 (r-r ')
From (Equation 6), when there is a positional shift amount r ′ in the X direction between two images, the peak position of the correlation strength image is shifted by −r ′. In addition, since the correlation calculation is performed using the phase component, the movement amount can be calculated even if there is a difference in brightness or contrast between the two images. When there is a positional shift amount in the X direction between two images, a peak occurs at a position of ΔG (pixel) from the center of the correlation strength image. For example, if there is a shift of 2 pixels in the X direction between two images, the composite image becomes a two-cycle wave. When this is subjected to inverse Fourier transform, a correlation intensity image is obtained, and a peak is generated at a position shifted by 2 pixels from the center. This ΔG (pixel) corresponds to a movement amount on the light receiving surface of the detector, and ΔG is converted into a movement amount Δx on the sample surface. Assuming that the diameter L of the light receiving surface of the detector, the magnification M of the transmission electron microscope on the light receiving surface, and the number of pixels Lm of the light receiving surface of the detector are shown in (Expression 7).
(Expression 7) Δx = ΔG (pixel) × L / Lm (pixel) / M
Δx is the amount of movement on the sample surface between the two images.
(2) Means for calculating the degree of coincidence Next, the amount of movement between images, magnification, and accuracy of the rotation angle will be described. In the correlation calculation using only the phase component, since only the phase is used mathematically, the peak appearing in the correlation strength is the δ peak. For example, if the image is shifted by 1.5 pixels between two images, the composite image becomes a wave of 1.5 cycles. When this is subjected to inverse Fourier transform, a δ peak appears at a position shifted by 1.5 pixels from the center of the correlation strength image, but since there is no 1.5 pixel, the value of the δ peak is assigned to the first pixel and the second pixel. Here, taking the center of gravity of the pixel having a high degree of coincidence and calculating the true δ peak position from the assigned value, the calculation result can be obtained with an accuracy of about 1/10 pixel. Further, since the correlation intensity image is a δ peak, the similarity between the two images is evaluated based on the peak height of the correlation intensity image. Assuming that image f1 (m, n) and peak height Peak (pixel) are given, the degree of coincidence (%) is shown in (Equation 8).
(Equation 8) Matching degree (%) = (Peak) / (m × n) × 100
For example, when the number of processed pixels is 128 pixels × 128 pixels and the peak is 16384 (pixels), the degree of coincidence = (16384) / (128 × 128) × 100 = 100 (%).
Using the calculation method described above, the control unit of the present embodiment has an image processing function for measuring the movement amount of at least two images, and can measure the movement amount of the optical microscope image and the electron microscope image. . Furthermore, it has an image moving function for moving images, and can automatically superimpose an optical microscope image and an electron microscope image based on the measured movement amount between images. FIG. 12 shows an example in which an optical microscope image is displayed on the electron microscope control GUI in a control GUI example that is an interface according to the embodiment of the present invention. Thereby, it is possible to superimpose the optical microscope image and the electron microscope image and to specify the area at the specified position.

FIG. 13 shows an example in which an optical microscope image is displayed on the electron microscope control GUI in the control GUI example according to the embodiment of the present invention. It is possible to superimpose an optical microscope and an electron microscope and to specify an area at a specified position.

DESCRIPTION OF SYMBOLS 1 ... Electron gun, 2 ... 1st irradiation lens coil, 3 ... 2nd irradiation lens coil, 4 ... 1st deflection coil, 5 ... 2nd deflection coil, 6 ... Objective lens coil, 7 ... Objective lens coil, 8 ... Objective Auxiliary lens coil, 9 ... first intermediate lens coil, 10 ... second intermediate lens coil, 11 ... first projection lens coil, 12 ... second projection lens coil, 13-23 ... excitation power source, 24-34 ... DAC, 35 ... Microprocessor, 36 ... Storage device, 37 ... Calculation device, 38 ... Monitor controller, 39 ... Monitor, 40-41 ... I / F, 42 ... Rotary encoder for switching magnification, 43 ... Rotary encoder for input, 44 ... Keyboard 45 ... RAM, 46 ... ROM, 47 ... ４７optical image capture interface, 48 ... image capture interface, 49 ... fluorescent screen 56 ... TV camera,

Claims

An electron source for irradiating the sample with an electron beam;
In an electron microscope equipped with a detector that detects electrons generated from the sample by irradiation of the electron beam,
An upper objective lens coil and a lower objective lens coil are provided above and below the sample,
An electron microscope characterized in that the polarities of the upper objective lens coil and the lower objective lens coil are reversed.
The electron microscope according to claim 1,
The electron microscope further comprises a control unit that controls to reverse the polarities of the upper objective lens coil and the lower objective lens coil to cancel the magnetic field.
The electron microscope according to claim 1,
In addition, it has an auxiliary lens built in the magnetic path,
An electron microscope characterized in that a current value of a lens coil is made constant during observation at extremely low magnification and during high-definition observation.
The electron microscope according to claim 2,
The control unit controls the electron microscope to automatically switch from the high-definition observation mode to the ultra-low magnification observation mode.
The electron microscope according to claim 4,
The control unit controls the objective aperture so that IN / OUT can be controlled from the optical axis, and when the automatic switching from the high-definition observation mode to the ultra-low magnification observation mode is performed, the objective aperture is adjusted from the optical axis to IN. Electron microscope that controls / OUT.
The electron microscope according to claim 2,
An electron microscope that has an interface for acquiring ultra-low magnification images and optical microscope images and can superimpose ultra-low magnification images and optical microscope images.
The electron microscope according to claim 5,
The control unit has an image processing function for measuring a movement amount of at least two or more images, and can measure the movement amount of an optical microscope image and an electron microscope image.
The electron microscope according to claim 7,
The control unit further has an image moving function for moving an image, and can automatically superimpose an optical microscope image and an electron microscope image based on a movement amount between measured images.