WO2020178994A1 - Microscope observation method, transmission-type microscope device, and scanning transmission-type microscope device - Google Patents

Microscope observation method, transmission-type microscope device, and scanning transmission-type microscope device Download PDF

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WO2020178994A1
WO2020178994A1 PCT/JP2019/008720 JP2019008720W WO2020178994A1 WO 2020178994 A1 WO2020178994 A1 WO 2020178994A1 JP 2019008720 W JP2019008720 W JP 2019008720W WO 2020178994 A1 WO2020178994 A1 WO 2020178994A1
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light source
dimensional
scanning
observation object
differential phase
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PCT/JP2019/008720
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French (fr)
Japanese (ja)
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國昭 永山
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Ｎ－Ｅｍラボラトリーズ株式会社
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/02—Details
    • H01J37/22—Optical or photographic arrangements associated with the tube
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes

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  • the present invention relates to a microscope observing method, a transmission microscope apparatus, and a scanning transmission microscope apparatus that obtain an intensity image and a differential phase image of an observation target by utilizing deflection of light and electrons when passing through a substance.
  • the phase difference between two waves having different phases is converted into intensity modulation due to wave interference, and the phase difference is analogized from the intensity image.
  • a phase reference is given. It is necessary to prepare a reference wave and a reference wave. For a simple method that uses a part of the signal wave as the reference wave, the size of the observable phase is limited to establish the "weak phase object approximation", and in the holography method that can overcome the limitation, Since a reference wave that is independent of the wave is required, it is inevitable to devise and complicate the device.
  • Non-Patent Documents 1 to 6 there has been proposed a method of obtaining the differential phase of an observation target object by utilizing the beam deflection by a substance without using the interference effect.
  • an observation object is irradiated with a convergent beam of electrons or X-rays while scanning, and deflection of electrons or X-rays caused by a change in the refractive index at the irradiation position is detected and imaged.
  • Non-Patent Documents 12 to 13 are also widely used as a four-dimensional scanning transmission electron microscope (4D-STEM, Non-Patent Documents 14 to 15).
  • the contrast represented by the integral detector is imaged as corresponding to the amount of movement of the center of gravity of the bright-field disk
  • COM-imaging-STEM or 4D-STEM The amount of movement of the center of gravity is obtained and imaged by performing integral calculation on the two-dimensional data of the bright-field disk.
  • Both COM-imaging-STEM and 4D-STEM are called the four-dimensional method because they acquire four-dimensional data as the sum of the two dimensions that scan on the observation object and the two dimensions of the bright-field disk.
  • Non-Patent Document 12 The two-dimensional method DPC-STEM and the four-dimensional method COM-imaging-STEM (4D-STEM) are said to give almost the same image, but the four-dimensional method (COM-imaging-STEM and 4D- STEM) is more quantitative (Non-Patent Document 12). Both of them are characterized by high resolution observation, and by using a convergent beam in the vicinity of 1 ⁇ , high resolution electric field observation of atoms and molecules in a crystal is performed (Non-Patent Documents 7 to 15).
  • the key of the present invention is to obtain a bright-field disk signal on the light source surface instead of the bright-field disk signal on the detection surface by changing the scanning transmission method to a transmission method in a four-dimensional microscope. .. In order to distinguish between the two, the terminology of bright-field disc on the detection surface and bright-field disc on the light source surface is used as necessary.
  • the four-dimensional scanning differential phase method using a convergent beam has the following problems.
  • the first problem is that in the scanning transmission method, the image observed on the detection surface is a diffraction image, and the image of the observation object cannot be directly observed during observation. It is not possible to respond promptly.
  • the second problem is the lengthening of the observation time and the enormous amount of digital storage capacity that accompany the acquisition of 4-dimensional observation data.
  • the third problem is that, especially in the case of an electron microscope, atomic resolution observation by DPC-STEM or 4D-STEM is limited to observation of metals and semiconductors that can withstand high-intensity electron beam irradiation, and biomolecules that are vulnerable to electron beam irradiation. It is not applicable to organic substances such as.
  • Non-Patent Documents 7 to 15 The principle itself of the method of observing a differential phase image of an observation object by scanning a convergent beam and deflecting an electron beam was proposed 40 years ago or more as seen in Non-Patent Document 1, but the above-mentioned problems Therefore, it has not been popularized in general microscope methods, and has been limited to applications to metals and semiconductors by a scanning electron microscope (Non-Patent Documents 7 to 15).
  • the present invention solves the problems of 4D-STEM, a four-dimensional microscope method of STEM using the above-mentioned convergent beam, and includes an object including an organic substance that is susceptible to electron beam fracture such as a biomolecule.
  • An object of the present invention is to provide a four-dimensional microscope observation method and a four-dimensional transmission microscope (4D-TEM) device capable of acquiring an intensity image and a differential phase image with high operability and high resolution and high sensitivity.
  • the microscope observation method includes an illumination step of irradiating an observation object in parallel with light emitted from a point light source from various angular directions, and a position where the observation object is deflected and conjugate with the point light source. 2D of the light source surface by performing an imaging step of condensing scattered light that has passed through the stopped diaphragm and forming a real image on the detection surface, and performing the illumination step and the imaging step while two-dimensionally scanning the point light source.
  • the scanning step of generating four-dimensional data consisting of the coordinates and the two-dimensional coordinates of the detection surface, and the integration of the center of gravity of the light source surface bright field disk at the two-dimensional coordinates of the light source surface are calculated in the four-dimensional data to obtain the observation object.
  • Another microscope observation method is an illumination step of irradiating light emitted from a point light source onto an observation object in parallel, and a diaphragm deflected by the observation object and arranged at a position conjugate with the point light source.
  • the imaging step of condensing the scattered light that has passed through to form a real image on the detection surface, the illumination step and the imaging step while two-dimensionally scanning the diaphragm, and the scanning two-dimensional coordinates and the detection surface 2 A two-dimensional differential phase image of the object to be observed by calculating the integration of the center of gravity of the light source surface bright-field disk at the light source surface two-dimensional coordinates in the scanning step of generating four-dimensional data consisting of dimensional coordinates and the four-dimensional data. And a differential phase image acquisition step of acquiring.
  • Another microscope observing method is an illumination step of irradiating the light emitted from the point light source to the observation object in parallel from various angular directions, a position deflected by the observation object, and a position conjugate with the point light source.
  • a differential phase image acquisition step of acquiring a two-dimensional differential phase image of the observation object by calculating the centroid integral of the bright field cane.
  • the transmission microscope apparatus includes an illumination optical system that irradiates an object to be observed with light emitted from a point light source in parallel from various angle directions, and a diaphragm at a position conjugate with the point light source for adjusting the light amount.
  • An imaging optical system that collects light that has passed through an object to be observed and forms a real image on the detection surface, and the illumination while two-dimensionally scanning the emission angle direction from the point light source.
  • the control unit that performs the imaging, the data generation unit that generates 4D data or 3D data composed of the 2D coordinates of the light source surface and the 2D coordinates of the detection surface, and the 4D data or 3D data are combined with the light source surface.
  • the illumination optical system may be configured by one or one group of lenses and one or one group of diaphragms.
  • the imaging optical system can be configured such that a diaphragm surface is arranged between at least two lenses.
  • parallel light is emitted instead of the convergent beam to form a real image on the detection surface, so that it is possible to reduce the dimension of four dimensions from the high dimension of three dimensions to the low dimension of three dimensions. Since the scanning range can be arbitrarily set including the dimension, it is possible to acquire the high-resolution and high-sensitivity intensity image and differential phase image of the observation object with good scannability through real image observation.
  • FIG. 3 is a schematic diagram of a diaphragm central shielding method in 4D-STEM for improving sensitivity and a light source sub-region scanning method in 4D-TEM. It is a schematic diagram of the one-dimensional light source scanning 3D-TEM method for dimension reduction.
  • A It is a schematic diagram of a TEM apparatus provided with a deflection coil for performing oblique illumination scanning. B.
  • FIG. 3 is an explanatory view of a full aperture scanning (one-dimensional scanning of the entire light source) using a laterally long slit diaphragm in the absence of electron beam deflection in 3D-TEM.
  • FIG. 3 is an explanatory diagram of a full aperture scanning (one-dimensional scanning over the entire light source) method using a laterally elongated slit diaphragm when electron beam is deflected in 3D-TEM.
  • FIG. 3 is an explanatory diagram of an aperture edge scanning (one-dimensional scanning of light source partial region) method using a laterally long slit diaphragm when there is no electron beam deflection in 3D-TEM.
  • FIG. 3 is an explanatory diagram of an aperture edge scanning (one-dimensional scanning of light source partial region) method using a laterally long slit diaphragm when electron beam is deflected in 3D-TEM. It is a figure which shows the variation of the aperture edge scanning (light source partial area one-dimensional scanning) method, (A) both ends both sides scanning method, (B) one end both sides scanning method, (C) both ends one side scanning method I, (D) both ends one side Scanning method II. It is a block diagram which shows the structural example of the transmission type microscope apparatus of 1st Embodiment of this invention. 3 is a flowchart showing a data processing method used in the first embodiment of the present invention.
  • Embodiment of the first group An observation method in which parallel light irradiation is used instead of convergent beam irradiation, two-dimensional scanning of a point light source or an aperture is performed, and a differential phase image is calculated and acquired from the obtained four-dimensional data.
  • Second group embodiment Observation method in which parallel light irradiation is used instead of convergent beam irradiation, one-dimensional scanning of a point light source or a diaphragm is performed, and a differential phase image is calculated and acquired from the obtained three-dimensional data.
  • Third group of embodiments parallel light irradiation is used instead of convergent beam irradiation, one-dimensional scanning is performed with a point light source or diaphragm limited to a scanning range near the aperture opening end, and differential phase is obtained from the obtained three-dimensional data.
  • FIG. 1A is a diagram schematically showing an image forming process of a conventional focused beam scanning diffraction image observation four-dimensional microscope (4D-STEM) using the above-described focused beam deflection.
  • 4D-STEM focused beam scanning diffraction image observation four-dimensional microscope
  • "four-dimensional" in a four-dimensional microscope is an expression of the basic idea of the present invention in which the two-dimensional coordinates of the light source surface and the two-dimensional coordinates of the detection surface are handled independently.
  • the first optical surface is a light source surface
  • the second optical surface is a diaphragm surface
  • the third optical surface is an object surface
  • the fourth optical surface is a detection surface.
  • the convergent beam deflected by the observation object passes through the third lens 103, and forms a detection surface bright-field disc, which is an image of the diaphragm, on the detection surface 114 installed on the back focal plane.
  • the movement of the detection surface bright field disk gives a differential phase, and a differential phase scanning image is obtained by scanning the light source position.
  • the image formation process by light is also a Fourier transform process by three lenses 101 to 103 connecting four optical surfaces, as will be seen below.
  • the light ⁇ (r) emitted from one point r s on the light source surface 111 on the front focal plane of the first lens 101 is subjected to an inverse Fourier transform (FT ⁇ ) when passing through the first lens 101.
  • FT ⁇ inverse Fourier transform
  • the action represented by 1 the action represented by the aperture function P (s) at the aperture, the action represented by the Fourier transform (FT) when passing through the second lens 102, and the probe function.
  • the convergent beam represented by p(r s ⁇ r) is applied to the object surface 113 represented by t(r).
  • the convergent beam deflected by the object is subjected to the action represented by the Fourier transform FT when passing through the third lens 103, and the detection represented by the following formula 1 is performed on the detection surface 114 placed on the back focal plane.
  • Form a bright field disc Form a bright field disc.
  • the imaging process of an object with light (beam) is also a Fourier transform process by three lenses that connect four optical surfaces.
  • the diaphragm function P(s) and the probe function p(r) are connected by Fourier transform.
  • the size of the detector necessary for quantifying the movement of the detection surface bright field disk is the size of the diameter of the detection surface bright field disk with the maximum movement width added considering the movement width of the detection surface bright field disk. Is the lower limit. Since the detection surface bright-field disk itself is an image of the diaphragm arranged on the diaphragm surface 112, its size is proportional to the size of the diaphragm, and the smaller the convergent beam is converged in order to improve the spatial resolution, the more inversely proportional the relationship is. The aperture at 2 is larger and the corresponding detector is also larger.
  • the observation accuracy of the differential phase is proportional to the spatial resolution of the detector. The required specifications of the detector depend on both the image spatial resolution and the differential phase observation accuracy to be obtained in this way.
  • ⁇ (r) is a phase distribution
  • n (r) is a refractive index distribution
  • l (r) is a thickness distribution
  • r is a two-dimensional spatial coordinate
  • ⁇ is a wavelength of a carrier wave.
  • “1” of (n(r) ⁇ 1) is the refractive index of air (to be exact, vacuum), and the change in the refractive index at the place with and without an object corresponds to the phase change. ing.
  • (n(r)-1)l(r) corresponds to the optical distance.
  • the distribution of the refractive index can be approximated to that of a small prism when viewed in a minute range.
  • the observation target is considered as a group of micro prisms, when a narrow portion is illuminated with convergent light (beam), the light (beam) is bent by the micro prisms in a direction different from the incident direction. This is the physical process of ray (beam) deflection that occurs when the focused light (beam) is applied to the observation object.
  • the simplest method of the deflection amount is to quantify the spatial movement associated with the deflection of the detection surface bright-field disc created on the detection surface by the convergent beam irradiated on the observation object.
  • the most simplified microscope optical system is a two-lens system composed of an objective lens and an eyepiece lens.
  • the light source surface 2 defined independently of the light source surface and the detection surface is used.
  • Dimensional coordinates and detection surface Since four-dimensional observation is performed based on four-dimensional coordinates expressed using two-dimensional coordinates, it is necessary to explicitly define the light source surface. Therefore, in the microscope observation method of the present embodiment, a three-lens system including a light source surface and a condenser lens (collector lens) as shown in FIG. 1A is adopted.
  • the three lenses have the same focal length f, and the seven elements having the same optical distance as the lenses have the same focal length interval. 3 lenses, 4 optical surfaces, and 6 focal point system combined by. Since the enlargement function of the microscope is not considered here, the imaging magnification is treated as 1. Further, in the present embodiment, the lens action is treated as a light converging effect when physically treated, and as a Fourier transform action when treated mathematically.
  • FIG. 1A illustrates a conventional differential phase image observation method in which a convergent beam is irradiated while being scanned in an optical system of a 3-lens / 4-optical surface system.
  • the optical field for revealing the detection MenAkira viewing disc denoted U STEM (r s, s d ) and using the frequency space coordinates s d of the real space coordinate r s and the detection surface of the light source plane.
  • the light source plane coordinate was placed first and the detection plane coordinate was placed second.
  • U STEM (r s, s d ) is a diffraction image two-dimensional function to expand the s d coordinate detection plane individual r s as a parameter, the object plane 2 dimensionally scanning the source point r s on conjugate light plane Only then will it be four-dimensional. Since the differential phase is represented by the movement of the bright-field disc on the detection surface due to the deflection of the convergent beam, the specific calculation is the detection as an s d coordinate intensity image (square detection image) formed by the optical field. MenAkira field disc function,
  • an object to be observed is irradiated with a convergent beam, the convergent beam is two-dimensionally scanned, and the total intensity of the diffraction image is acquired by a wide-angle detector placed on a detection surface.
  • the illumination light strikes a narrow area of the observation target, so that the degree of deflection can be specified locally.
  • the illumination light is irradiated to a wide area of the observation object and is deflected in all directions, so the degree of local deflection on the observation object is specified, and the differential phase is dependent on the location. It has been considered impossible to extract.
  • FIG. 1A is an image diagram of a conventional scanning four-dimensional method (4D-STEM) using a convergent beam
  • FIG. 1B is a schematic view of parallel illumination light emitted from a large number of point light sources according to the present invention.
  • FIG. 1B conceptually shows the superposition of different individual point light source experiments.
  • 1A and 1B are the objects to be mirrored, which are between the scanning transmission electron microscope (STEM) using the convergent illumination light and the transmission electron microscope (TEM) using the parallel illumination light, which have already been found in the electron microscope.
  • STEM scanning transmission electron microscope
  • TEM transmission electron microscope
  • each light beam is converged by the lens 101 and the lens 102 to one point on the object surface 113, scattered by the observation object, diffracted by the lens 103, and spread on the two-dimensional coordinates of the detection surface 114.
  • the image is observed as a brightfield disc.
  • the bright-field disk is shown in FIG. 2.
  • the solid line shown in FIG. 1A shows how the light beam is locally deflected by the observation object, and the wavy line shows how it goes straight when there is no observation object. .. That is, FIG. 1A clearly shows that the detection surface bright-field disk moves on the detection surface 114 as the convergent beam is deflected by the observation object. This amount of movement corresponds to the amount of differential phase.
  • FIG. 1B is a reverse light ray diagram of FIG. 1A, and is a diagram of a configuration in which parallel illumination light, each of which has an inclination with respect to the optical axis, is irradiated onto an observation object from a large number of points on the light source surface.
  • FIG. 1B is an image diagram in which the light source surface and the detection surface in FIG. 1A are replaced with each other and then the images are reversed to the left and right. It is shown that light is emitted from three different point light sources.
  • the ray diagram in this case also differs depending on the presence or absence of the observation object on the object surface 22, and the state is shown by the solid line and the broken line in FIG. 1B.
  • the differential phase amount can be obtained at each point on the observation object.
  • Fig. 2 shows the movement of both the detection surface bright-field disk of the 4D-STEM method and the corresponding light source surface bright-field disk of the 4D-TEM method due to electron beam deflection in a four-dimensional reciprocity. ..
  • the frequency space coordinates s d are s s of the detection surface (detection plane)
  • the real space coordinates r s of the light source surface (source plane) to r d are changed respectively.
  • the deflection of the electron beam so appears as the movement of the light source MenAkira field disc observed virtually corresponds to the individual r d, the derivative phase amount, the light source MenAkira field disc function is expanded in two dimensions s s coordinates securing the r d
  • the point light source In the normal TEM parallel light irradiation method, the point light source is placed on the optical axis, but in the 4D-STEM parallel light irradiation method, it is emitted from a point away from the optical axis, as in the convergent beam irradiation four-dimensional method.
  • the electron beam is two-dimensionally scanned.
  • the object to be observed is illuminated from a point distant from the optical axis, that is, light (beam) having an inclination with respect to the optical axis is used instead of irradiation of light (beam) parallel to the normal optical axis.
  • irradiating hereinafter, referred to as “oblique illumination”
  • a real image observed on the detection surface is recorded.
  • the light source point is two-dimensionally scanned, and each time the observation image is accumulated in the data storage unit provided in the microscope apparatus.
  • the data is developed into four-dimensional data on the four-dimensional coordinate which is a combination of the two-dimensional coordinate of the light source surface and the two-dimensional coordinate of the real image detecting surface.
  • the four-dimensional image obtained by the convergent light (beam) irradiation has a probe function p(r) of the irradiation light represented by the following formula 3 and an object transmission function t(r) represented by the following formula 4. It is expressed by the following Equation 5.
  • the probe function p (r) and the object transmission function t (r) are both complex numbers.
  • the diaphragm function P(s) in the above formula 6 is expressed by the following formula 7, and is connected to the irradiation light probe function by the Fourier transform FT.
  • T (s) is expressed by the following mathematical formula 8 and is connected to the object transmission function by the Fourier transform. Both P(s) and T(s) are complex numbers.
  • the differential phase amounts in the four-dimensional method by the convergent light (beam) irradiation and the four-dimensional method by the parallel light (beam) irradiation are expressed by the mathematical expressions 9 and 10 using the above mathematical expressions 2 to 8, respectively.
  • Equations 9 and 10 the numerator represents the first moment and the denominator represents the area corresponding to the signal strength. Further, division corresponds to the center of gravity, and such calculation is called center of gravity integration.
  • the above equation 9 for the s d centroid includes both the derivative ⁇ t (r) of the phase component of the transmission coefficient and the derivative ⁇ p (r) of the phase component of the probe function.
  • the differential code and the like are slightly different, this is the same as the mathematical formula first derived in the world by Waddell et al. (see Non-Patent Document 1).
  • the expression of the s s center of gravity shown in the above equation 10 was discovered by the present inventor, and this application is the first in the world.
  • the differential used in the parallel light irradiation four-dimensional microscope observation method of the present invention means a two-dimensional first-order differential, that is, the gradient shown in the following formula 11, and ⁇ t (r) is represented by the following formula 12. ..
  • the differential phase of the observation object can be obtained by performing the s center of gravity calculation shown in Equations 9 and 10 for both the four-dimensional microscope data by the focused light irradiation and the four-dimensional microscope data by the parallel light irradiation.
  • the line integral operation shown in Equation 15 below may be performed according to the formula for obtaining the potential from the gradient.
  • Is a straight line (x f , y) parallel to, and the end point is the path to be the desired r f (x f , y f ) point. Equation 15 also shows that it is sufficient to have two orthogonal differential phase components to restore the phase.
  • 4D-STEM uses a large shielding plate in the center of the aperture, and 4D-TEM scans the light source. Is limited to the peripheral portion of the diaphragm, specifically, the aperture end scanning is performed. According to this method, the electron beam resources of the portion that does not need to be seen can be concentrated in the signal portion corresponding to the peripheral portion of the diaphragm that carries the signal, so that the signal-to-noise ratio is increased and the sensitivity is improved.
  • 4D-STEM Even with the same electron beam uneven irradiation, there is a difference between 4D-STEM and 4D-TEM, and 4D-STEM sacrifices resolution because it blocks the diaphragm.
  • 4D-TEM the resolution can be set freely because it is realized only by controlling the light source scanning range without giving shape changes such as shielding to the diaphragm.
  • the 4D-TEM method of partial scanning of the light source overcomes the fundamental problem in the microscope, the conflict of resolution and sensitivity for the first time.
  • the kicking of the electron beam occurs only at the slit end orthogonal to the longitudinal direction, so that the differential phase in that direction can be obtained. It will be.
  • the dimension reduction can also be applied to the partial scanning method described in the sensitivity improvement, and the observation time can be further shortened by reducing the scanning range.
  • FIG. 5 schematically shows the basic form of this method, in which one-dimensional scanning of the light source is performed in the direction orthogonal to the lateral slit.
  • the scanning area is set so as to cover the width of the slit up to the maximum value of the electron beam deflection of the observation target and scan the entire area of the slit.
  • the slit type light source surface bright field disc corresponding to the slit (the expression disc is used as a terminology, but in the case of one-dimensional scanning, what is actually observed is on the light source scanning line orthogonal to the longitudinal direction.
  • FIG. 6 schematically shows a slit-type light source surface clear-field disk, slits, and a sequence of light source points when the electron beam is deflected, and one-dimensional scanning of the light source is performed in a direction orthogonal to the horizontally long slit as in FIG. To do.
  • the scanning area is set so as to cover the width of the slit up to the maximum value of the electron beam deflection of the observation target and scan the entire area of the slit.
  • the deflection occurs in the negative direction of the y-axis, and the y-direction center of gravity of the point sequence moves downward from the origin in proportion to the deflection. This gives a negative differential phase.
  • Fig. 7 the relationship between the slit type light source surface bright field disk and the slit is the same as in Fig. 5, but the light source scanning range is limited to the vicinity of the upper and lower ends of the slit.
  • the signal at the central portion of the slit is cut, if there is no electron beam deflection, the y-direction center of gravity of the data obtained by this partial scanning is the same as the origin and does not move, as in FIG.
  • the relationship between the slit type light source surface bright field disk and the slit is the same as in FIG. 6, but the light source scanning range is limited to the vicinity of the upper and lower ends of the slit.
  • the y-direction center of gravity of the data obtained by this partial scan moves downward.
  • the amount of movement of the center of gravity observed at this time is the same as the amount of movement of the center of gravity observed by the whole-area scanning method shown in FIG. Nevertheless, since the total electron dose of the whole area scanning shown in FIG. 6 is directed to a narrow scanning range, the electron dose at each scanning point increases and the shot noise peculiar to electron beam observation decreases relatively, and the signal-to-noise ratio. Will improve. That is, the sensitivity is improved.
  • various embodiments configured according to the conventional principle invention will be described.
  • the first embodiment faithfully executes the four-dimensional transmission electron microscope principle described above, and an illumination step of irradiating an object to be observed with light emitted from a point light source in parallel from an oblique direction at an arbitrary angle.
  • a scanning step in which the illumination step and the imaging step are performed while dimensionally scanning to generate four-dimensional data composed of two-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface, and the four-dimensional data are obtained from the two-dimensional coordinates of the light source surface.
  • a differential phase image acquisition step of acquiring a two-dimensional differential phase image of the observation object by calculating the barycentric integral in.
  • FIG. 10 is a block diagram showing a configuration example of the transmission microscope apparatus 1 of this embodiment.
  • an optical system 2 having a diaphragm, an imaging unit 3 for detecting a formed two-dimensional image, a control unit 4 for controlling light source scanning, and the like, This can be performed by the transmission microscope apparatus 1 including the data processing unit 5 that stores, stores, and calculates the four-dimensional image data.
  • the optical system 2 is provided with a light source and an imaging system using a plurality of lenses that realize the optical system shown in FIGS. 1 and 2, for example.
  • the arrangement of the aperture and the observation object is different between the convergent beam irradiation four-dimensional method proposed by Waddell et al. Shown in FIG. 1A and the parallel light (beam) irradiation four-dimensional method of the present invention shown in FIG. 1B.
  • the basic configuration of the optical system 2 of the transmission microscope apparatus 1 is the same as that of the convergent beam irradiation four-dimensional method proposed by Waddell et al.
  • a two-dimensional detector can be used for the imaging unit 3.
  • the four-dimensional microscope of the present embodiment is the same as the electron microscope having the STEM function in terms of light source scanning, but the original method of scanning the observation object on the object surface with the convergent beam is a parallel light (beam) irradiation. It is realized by changing the method of scanning the irradiation direction of.
  • the microscope observation method of the present embodiment is a parallel light illumination four-dimensional method, and since the real image is the primary observation target, the intensity image and the differential phase image of the observation target are manipulated. It can be acquired well. Further, the transmission microscope apparatus 1 of the present embodiment can be realized by utilizing the STEM function of the STEM apparatus and using it as a TEM or by adding the STEM function to the TEM.
  • Step 2 Integration operation> Using 4-dimensional data obtained in step 1, performs area integration on the two-dimensional s s coordinates for individual r d, obtain intensity image. In parallel, using a four-dimensional data obtained in step 1, perform the centroid integration on the two-dimensional s s coordinates for individual r d, i.e. s s coordinates a moment calculated on the strength it Divide by the image to obtain the differential phase image.
  • the diaphragm is not a normal circular shape, but a slit extending in a straight line in a horizontally long shape. Then, the scanning of the point light source or the slit is performed in one dimension orthogonal to the slit longitudinal direction, and in the scanning step of generating three-dimensional data, as shown in FIGS. The entire area of the opening is scanned symmetrically on both sides from a position beyond the opening end of the slit covering the slit. This is called the full aperture scanning 3D-TEM method. This reduction in dimension improves observation efficiency.
  • the microscope apparatus and the data processing method are the same as those in the first embodiment because there is no difference from the first embodiment except that the scanning method is changed from two-dimensional scanning to one-dimensional scanning.
  • the description of the second and subsequent embodiments the description of the same configuration as that of the first embodiment will be omitted.
  • the microscope apparatus and the data processing method are the same as those in the first embodiment. is there.
  • the slit extends horizontally in a straight line. Then, in the scanning step of scanning the point light source in one dimension and generating three-dimensional data, the scanning region of the point light source is at least one of both ends in the scanning direction excluding the central portion of the opening of the slit. Limited to one side. This is called the aperture edge scanning 3D-TEM method.
  • the scanning region is defined over both sides in the scanning direction with respect to at least one of the pair of opening edges in the slit in a plan view seen from the optical axis direction orthogonal to the slit, and the scanning region
  • the total sum is half of the maximum expected differential phase amount.
  • the scanning region is evenly defined across both sides in the scanning direction with respect to at least one of the pair of aperture edges.
  • the scanning area is evenly defined across both sides of the scanning direction with respect to both open edge edges.
  • FIG. 9 is an example of a variation of both-ends both-sides opening-end scanning method 61 (FIG. 9A) shown in FIGS. 7 and 8.
  • the scanning region may be evenly defined across both sides of one open edge with respect to one open edge, as in the single-ended bilateral open-end scanning method 62 shown in FIG. 9B.
  • the double-ended single-sided open end scanning I method 63 shown in FIG. 9C and the double-ended single-sided open end running II method 64 shown in FIG. May be evenly defined on each side.
  • the relationship among the four quantities, the electron beam deflection angle of the observation object, the electron beam irradiation amount, the spatial resolution, and the differential phase resolution (corresponding to the observation accuracy of the image contrast by the observation accuracy of the deflection angle) is generally the full aperture scan (the entire light source area).
  • Scanning) 4D-TEM and open edge scanning (light source partial area scanning) 4D-TEM is different, and partial electron beam scanning is performed in electron microscopic observation of biological molecules such as proteins and DNA that have an upper limit on electron beam irradiation.
  • Open edge scanning 4D-TEM is advantageous.
  • the sensitivity improvement for the 3D-TEM method using a horizontally long slit for dimension reduction will be described in detail.
  • FIG. 5 and FIG. 6 are schematic diagrams showing a case without electron beam deflection (A) and a case with electron beam deflection (B) in the full aperture scanning 3D-TEM experiment.
  • a slit diaphragm located at a position conjugate with the light source is projected on the light source surface, and the light source scanning points are displayed on the light source surface s y axis for schematically performing one-dimensional scanning.
  • the shaded area in FIG. 6 shows the image when the electron beam is deflected at the local r of the observation object, that is, when ⁇ (r) ⁇ 0, the electron beam reaches the detector without being blocked by the slit diaphragm and the image is observed. It is a range. Above the slit in FIG. 6, an electron beam bypassing the slit due to deflection reaches the detector and is observed, and conversely, in FIG. 5, the observed electron beam is blocked by the slit and is not observed.
  • the aperture range is [-L/2, L/2] (L is an integer), but the scanning range has a margin of l/4 [-L/2-l/4, L/2+l/4] ( Both L and l are integers), and the scan width is L+l/2. It is important that the magnitude
  • FIGS. 7 and 8 show schematic diagrams in the case where there is no electron beam deflection in the aperture edge scanning 3D-TEM (A) and the case (B).
  • the difference from the full aperture scanning 3D-TEM in FIGS. 5 and 6 is that the scanning range is limited to the vicinity of the aperture end. Therefore, the scanning range is [-L/2-l/4, -L/2+ l/4] and [L/2-l/4, L/2+l/4] are bisected and narrowed (both L and l are integers), and the total scan width is l.
  • l is extremely small compared to L, so when the total irradiation electron dose is limited, open-edge scanning 3D-TEM, which can concentrate the electron beam in a narrow scanning range l, can reduce the noise derived from shot noise, resulting in high. Sensitivity is possible.
  • ⁇ (r) has the same dimension as s of 3D data,
  • 2 , and the s y axis of s is defined by the following equations (20) to (21). .. s y (2 ⁇ / ⁇ ) ⁇ (a/f) (20) (A: slit stop s y- axis actual size, f: lens focal length) Since a / f is a direction seen from the detector aperture coordinate s y, it can be seen that (19) [Delta] n in formula (r) corresponds to the electron beam deflection angle of s y-axis.
  • the slit end coordinates s yo are as follows using Eq. (20), and a o / f is the so-called numerical aperture of the objective lens.
  • s yo (2 ⁇ / ⁇ ) ⁇ (a o /f) (21)
  • the numerical aperture a o / f is also connected to the physical resolution ⁇ p of the microscope in the following relationship.
  • ⁇ p ⁇ /(a o /f) (22)
  • the fluctuation (standard deviation) of the position of the center of gravity derived from shot noise in the two 3D-TEM methods is calculated.
  • the magnitude of this amount determines the sensitivity of the method.
  • a large amount of electron beam irradiation is required as shown below and it cannot be applied to biomolecules.
  • the electron beam input in the shaded areas shown in FIGS. 5 to 8 is uniform, and the number of electrons coming to each point (each pixel) is a certain average value (this is the same regardless of the pixel position. (Meaning uniform input) around the Poisson distribution (shot noise assumption).
  • the center of gravity of the shaded area is originally the center of the rectangle, but it fluctuates from the center position due to shot noise.
  • This fluctuation, to be exact, the standard deviation becomes smaller as the total number of electron beams coming to the shaded area increases.
  • the standard deviation is expressed by equation (23) in the case of full aperture scanning 3D-TEM in FIGS. 5 and 6, and by equation (24) in the case of aperture edge scanning 3D-TEM in FIGS. 7 and 8. .
  • the ratio between the scanning range of the light source and the average deflection angle value is required.
  • the scanning range is 2a o / f shown in Eq. (21)
  • the average deflection angle is the average value of ⁇ n (r).
  • a o /f is related to the physical resolution as a numerical aperture.
  • Microscope device 2 10, 100 Optical system 3 Imaging unit 4 Control unit 5 Data processing unit 11 to 13, 101 to 103 Lens 21, 111 Light source surface 22, 113 Object surface 23, 112 Aperture surface 24, 114 Detection surface 26 Deflection Coil 31 Detection surface without movement of center of gravity / light source surface bright field disk 32 Detection surface with movement of center of gravity / light source surface bright field disk 41 4 Partial scanning area of light source in D-TEM 42 4 Circular diaphragm used for D-STEM 43 4D-STEM Electron beam shielding plate 51 used for a diaphragm aperture 52 Horizontal slit (aperture) 60 one-dimensional scanning direction 61 both ends both sides opening end scanning method 62 one end both sides opening end scanning method 63 both ends one side opening end scanning I method 64 both ends one side opening end scanning II method

Abstract

The present invention provides a microscope observation method and a transmission-type microscope device with which it is possible to solve the problem of a conventional four-dimensional microscope method in which a convergent beam is used, and to easily acquire a high-resolution and high-sensitivity intensity image and differential phase image of an object under observation.　A microscope observation method and a transmission-type microscope device having: an illumination step for performing parallel irradiation, from various angular directions, of an object under observation with light emitted from a point light source; an imaging step for collecting scattered light, which has been deflected by the object under observation and has passed through an aperture disposed at a position conjugate with the point light source, and forming an actual image on a detection surface; a scanning step for performing the illumination step and the imaging step while two-dimensionally scanning the point light source to generate four-dimensional data comprising light source surface two-dimensional coordinates and detection surface two-dimensional coordinates; and a differential phase image acquisition step for performing a center-of-gravity integral calculation in the light source surface two-dimensional coordinates on the four-dimensional data to acquire a two-dimensional differential phase image.

Description

顕微鏡観察方法、透過型顕微鏡装置、および走査透過型顕微鏡装置Microscope observation method, transmission microscope apparatus, and scanning transmission microscope apparatus
　本発明は、物質を通過する際の光や電子の偏向を利用して観察対象物の強度像及び微分位相像を取得する顕微鏡観察方法、透過型顕微鏡装置、および走査透過型顕微鏡装置に関する。 The present invention relates to a microscope observing method, a transmission microscope apparatus, and a scanning transmission microscope apparatus that obtain an intensity image and a differential phase image of an observation target by utilizing deflection of light and electrons when passing through a substance.
　従来、顕微鏡による位相情報の取得には、主に干渉効果を利用した方法が用いられてきた。例えば、光学顕微鏡では、シュリーレン法、ゼルニケ位相差顕微鏡法及びノマルスキー微分干渉顕微鏡法などが知られている。また、電子顕微鏡では、焦点はずしを用いるデフォーカス法や位相板を用いる位相差電子顕微鏡などが知られている。 Conventionally, methods that mainly use the interference effect have been used to acquire phase information with a microscope. For example, as optical microscopes, Schlieren method, Zernike phase contrast microscope method, Nomalsky differential interference contrast microscope and the like are known. Further, as electron microscopes, a defocus method using defocusing, a phase contrast electron microscope using a phase plate, and the like are known.
　これらの手法はいずれも、位相の異なる２つの波の位相差を、波の干渉に伴う強度変調に変換し、強度像から位相差を類推するものであり、信号波の他に位相基準を与える参照用の波、参照波を用意する必要がある。参照波として、信号波の一部を利用する簡便法については、「弱位相物体近似」を成立させるために観測可能な位相の大きさが制限され、その限界を克服できるホログラフィー法などでは、信号波とは別に独立した参照波が必要となるため、装置の工夫や複雑化が避けられない。 In all of these methods, the phase difference between two waves having different phases is converted into intensity modulation due to wave interference, and the phase difference is analogized from the intensity image. In addition to the signal wave, a phase reference is given. It is necessary to prepare a reference wave and a reference wave. For a simple method that uses a part of the signal wave as the reference wave, the size of the observable phase is limited to establish the "weak phase object approximation", and in the holography method that can overcome the limitation, Since a reference wave that is independent of the wave is required, it is inevitable to devise and complicate the device.
　一方電子線やＸ線分野では、干渉効果を用いず、物質によるビームの偏向を利用して観察対象物の微分位相を求める手法が提案されてきた（非特許文献１～６参照）。これらの手法では、電子又はＸ線の収束ビームを、走査しながら観察対象物に照射し、その照射位置で生じる屈折率変化に起因する電子又はＸ線の偏向を検出して画像化している。特に近年走査透過型電子顕微鏡（STEM）における応用は目覚しく、微分干渉コントラスト走査透過型電子顕微鏡（DPC-STEM、非特許文献７～１１）や質量中心画像走査透過型電子顕微鏡（COM-imaging-STEM、非特許文献１２～１３）さらに4次元走査透過型電子顕微鏡（4D-STEM、非特許文献１４～１５）などと呼ばれ盛んに用いられている。 On the other hand, in the field of electron beams and X-rays, there has been proposed a method of obtaining the differential phase of an observation target object by utilizing the beam deflection by a substance without using the interference effect (see Non-Patent Documents 1 to 6). In these methods, an observation object is irradiated with a convergent beam of electrons or X-rays while scanning, and deflection of electrons or X-rays caused by a change in the refractive index at the irradiation position is detected and imaged. Particularly in recent years, its application in scanning transmission electron microscopes (STEMs) has been remarkable, such as differential interference contrast scanning transmission electron microscopes (DPC-STEM, Non-Patent Documents 7 to 11) and mass center image scanning transmission electron microscopes (COM-imaging-STEM). Non-Patent Documents 12 to 13), and is also widely used as a four-dimensional scanning transmission electron microscope (4D-STEM, Non-Patent Documents 14 to 15).
　屈折率変化は、位相変化、即ち微分位相の大きさに対応するため、非特許文献１～１５に記載の技術では、収束ビームの偏向度合いを定量的に取り出すために、収束ビームによる電子線回折像（一般に明視野円盤（bright-field disk）と呼ばれている）の重心移動を観測し、観察対象物の微分位相走査像を構成している。２次元観察法であるDPC-STEMでは、積分検出器に表象されるコントラストを明視野円盤重心移動量に対応するとして画像化、４次元観察法であるCOM-imaging-STEMや4D-STEMでは、明視野円盤の２次元データに対し積分計算を行うことで重心移動量を求め画像化している。COM-imaging-STEMと4D-STEM共に観察対象物上を走査する２次元と明視野円盤の２次元の和として４次元データを取得するので４次元法と呼ばれている。2次元法であるDPC-STEMと4次元法であるCOM-imaging-STEM（4D-STEM）は、ほぼ同等の画像を与えるといわれているが、４次元法（COM-imaging-STEMや4D-STEM）がより定量的である（非特許文献１２）。両者共に高分解能観察に特徴があり、１Å近辺の収束ビームを用いることで、結晶中の原子や分子の高分解能電場観察などを行っている（非特許文献７～１５）。
用語について：本発明の鍵は、4次元顕微鏡において、走査透過手法を透過手法に変えることで、検出面上の明視野円盤信号に代えて光源面上の明視野円盤信号を取得することである。両者を区別するため必要に応じ検出面上明視野円盤、光源面上明視野円盤の用語法を採用する。 Since the change in the refractive index corresponds to the phase change, that is, the magnitude of the differential phase, in the techniques described in Non-Patent Documents 1 to 15, in order to quantitatively extract the degree of deflection of the convergent beam, electron beam diffraction by the convergent beam is performed. The movement of the center of gravity of the image (generally called a bright-field disk) is observed to form a differential phase scanning image of the observation object. In the two-dimensional observation method DPC-STEM, the contrast represented by the integral detector is imaged as corresponding to the amount of movement of the center of gravity of the bright-field disk, and in the four-dimensional observation method COM-imaging-STEM or 4D-STEM, The amount of movement of the center of gravity is obtained and imaged by performing integral calculation on the two-dimensional data of the bright-field disk. Both COM-imaging-STEM and 4D-STEM are called the four-dimensional method because they acquire four-dimensional data as the sum of the two dimensions that scan on the observation object and the two dimensions of the bright-field disk. The two-dimensional method DPC-STEM and the four-dimensional method COM-imaging-STEM (4D-STEM) are said to give almost the same image, but the four-dimensional method (COM-imaging-STEM and 4D- STEM) is more quantitative (Non-Patent Document 12). Both of them are characterized by high resolution observation, and by using a convergent beam in the vicinity of 1Å, high resolution electric field observation of atoms and molecules in a crystal is performed (Non-Patent Documents 7 to 15).
Regarding terms: The key of the present invention is to obtain a bright-field disk signal on the light source surface instead of the bright-field disk signal on the detection surface by changing the scanning transmission method to a transmission method in a four-dimensional microscope. .. In order to distinguish between the two, the terminology of bright-field disc on the detection surface and bright-field disc on the light source surface is used as necessary.
　しかしながら、収束ビームを用いた４次元の走査型微分位相法には、以下に示す問題点がある。第１の問題点は、走査透過法は、検出面で観測される像が回折像で、観察時には観察対象物の画像を直接観察できないため、各種光学調整を行うための操作が必要な場合に速やかに対応することができないことである。 However, the four-dimensional scanning differential phase method using a convergent beam has the following problems. The first problem is that in the scanning transmission method, the image observed on the detection surface is a diffraction image, and the image of the observation object cannot be directly observed during observation. It is not possible to respond promptly.
　第２の問題点は、4次元の観測データを取得することに伴う観測時間の長大化とデジタル記憶容量の膨大化である。 The second problem is the lengthening of the observation time and the enormous amount of digital storage capacity that accompany the acquisition of 4-dimensional observation data.
　第３の問題点は、特に電子顕微鏡の場合、DPC-STEMや4D-STEMによる原子分解能観察が、高強度の電子線照射に耐える金属や半導体の観察に限定され、電子線照射に弱い生体分子などの有機物に適用できないことである。 The third problem is that, especially in the case of an electron microscope, atomic resolution observation by DPC-STEM or 4D-STEM is limited to observation of metals and semiconductors that can withstand high-intensity electron beam irradiation, and biomolecules that are vulnerable to electron beam irradiation. It is not applicable to organic substances such as.
　収束ビームの走査と電子線の偏向により観察対象物の微分位相像を観察する手法の原理そのものは、非特許文献１に見られるように４０年以上前に提案されていたが、前述した問題点から一般の顕微鏡法では普及せず、走査型電子顕微鏡による金属、半導体への応用（非特許文献７～１５）などに限定されてきた。 The principle itself of the method of observing a differential phase image of an observation object by scanning a convergent beam and deflecting an electron beam was proposed 40 years ago or more as seen in Non-Patent Document 1, but the above-mentioned problems Therefore, it has not been popularized in general microscope methods, and has been limited to applications to metals and semiconductors by a scanning electron microscope (Non-Patent Documents 7 to 15).
　そこで、本発明は、前述した収束ビームを利用したSTEMの４次元顕微鏡手法、4D-STEMの問題点を解決し、生体分子のような電子線破破壊を受けやすい有機物までをも含む対象物の強度像及び微分位相像を操作性良く高分解能かつ高感度で取得することができる4次元顕微鏡観察方法及び4次元透過型顕微鏡（4D-TEM）装置を提供することを目的とする。 Therefore, the present invention solves the problems of 4D-STEM, a four-dimensional microscope method of STEM using the above-mentioned convergent beam, and includes an object including an organic substance that is susceptible to electron beam fracture such as a biomolecule. An object of the present invention is to provide a four-dimensional microscope observation method and a four-dimensional transmission microscope (4D-TEM) device capable of acquiring an intensity image and a differential phase image with high operability and high resolution and high sensitivity.
　本発明に係る顕微鏡観察方法は、点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明工程と、前記観察対象物で偏向され、前記点光源と共役な位置に配置された絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、前記点光源を２次元走査しながら前記照明工程と前記結像工程を行い、光源面２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、前記４次元データにおいて、前記光源面２次元座標における光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する。 The microscope observation method according to the present invention includes an illumination step of irradiating an observation object in parallel with light emitted from a point light source from various angular directions, and a position where the observation object is deflected and conjugate with the point light source. 2D of the light source surface by performing an imaging step of condensing scattered light that has passed through the stopped diaphragm and forming a real image on the detection surface, and performing the illumination step and the imaging step while two-dimensionally scanning the point light source. The scanning step of generating four-dimensional data consisting of the coordinates and the two-dimensional coordinates of the detection surface, and the integration of the center of gravity of the light source surface bright field disk at the two-dimensional coordinates of the light source surface are calculated in the four-dimensional data to obtain the observation object. A differential phase image acquisition step of acquiring a two-dimensional differential phase image.
　本発明に係る他の顕微鏡観察方法は、点光源から射出された光を観察対象物に平行照射する照明工程と、前記観察対象物で偏向され、前記点光源と共役な位置に配置された絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、前記絞りを２次元走査しながら前記照明工程と前記結像工程を行い、走査２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、前記４次元データにおいて、前記光源面２次元座標における光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する。 Another microscope observation method according to the present invention is an illumination step of irradiating light emitted from a point light source onto an observation object in parallel, and a diaphragm deflected by the observation object and arranged at a position conjugate with the point light source. The imaging step of condensing the scattered light that has passed through to form a real image on the detection surface, the illumination step and the imaging step while two-dimensionally scanning the diaphragm, and the scanning two-dimensional coordinates and the detection surface 2 A two-dimensional differential phase image of the object to be observed by calculating the integration of the center of gravity of the light source surface bright-field disk at the light source surface two-dimensional coordinates in the scanning step of generating four-dimensional data consisting of dimensional coordinates and the four-dimensional data. And a differential phase image acquisition step of acquiring.
　本発明に係る他の顕微鏡観察方法は、点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明工程と、前記観察対象物で偏向され、前記点光源と共役な位置に配置された横長スリットである絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、前記点光源からの光又は前記絞りの走査を横長スリットの長手方向に直交した１次元で行い、光源面1次元座標と検出面2次元座標からなる３次元データ生成する走査工程と、前記３次元データにおいて、前記光源面1次元座標における光源面1次元明視野円盤（明視野杖）の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する。 Another microscope observing method according to the present invention is an illumination step of irradiating the light emitted from the point light source to the observation object in parallel from various angular directions, a position deflected by the observation object, and a position conjugate with the point light source. An image forming step of forming a real image on the detection surface by collecting scattered light that has passed through a diaphragm which is a horizontally elongated slit, and scanning the light from the point light source or the diaphragm in the longitudinal direction of the horizontally elongated slit. A scanning step of generating three-dimensional data consisting of one-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface, which is performed in one-dimensional orthogonal directions, and a one-dimensional bright-field disk of the light source surface at the one-dimensional coordinates of the light source surface in the three-dimensional data. And a differential phase image acquisition step of acquiring a two-dimensional differential phase image of the observation object by calculating the centroid integral of the bright field cane.
　本発明に係る透過型顕微鏡装置は、点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明光学系と、光量を調整するために前記点光源と共役な位置に絞りを配置する絞り部と、観察対象物を通過した光を集光して検出面に実像を結像させる結像光学系と、前記点光源からの射出角度方向を２次元走査しながら前記照明と前記結像を行う制御部と、光源面２次元座標と検出面２次元座標からなる４次元データまたは３次元データを生成するデータ生成部と、前記４次元データまたは３次元データを、前記光源面２次元または1次元座標における光源面明視野円盤の重心積分を計算するデータ処理部と、を有し、前記観察対象物の２次元強度像および微分位相像を得るものである。
　本発明の透過型顕微鏡装置は、前記照明光学系が、１枚又は１群のレンズ及び１枚又は１群の絞りにより構成されていてもよい。
　また、本発明の透過型顕微鏡装置は、前記結像光学系を、少なくとも２枚のレンズの間に絞り面が配置された構成とすることができる。 The transmission microscope apparatus according to the present invention includes an illumination optical system that irradiates an object to be observed with light emitted from a point light source in parallel from various angle directions, and a diaphragm at a position conjugate with the point light source for adjusting the light amount. An imaging optical system that collects light that has passed through an object to be observed and forms a real image on the detection surface, and the illumination while two-dimensionally scanning the emission angle direction from the point light source. The control unit that performs the imaging, the data generation unit that generates 4D data or 3D data composed of the 2D coordinates of the light source surface and the 2D coordinates of the detection surface, and the 4D data or 3D data are combined with the light source surface. It has a data processing unit that calculates the integration of the center of gravity of the light source surface bright-field disk in two-dimensional or one-dimensional coordinates, and obtains a two-dimensional intensity image and a differential phase image of the observation object.
In the transmission microscope apparatus of the present invention, the illumination optical system may be configured by one or one group of lenses and one or one group of diaphragms.
Further, in the transmission microscope apparatus of the present invention, the imaging optical system can be configured such that a diaphragm surface is arranged between at least two lenses.
　本発明によれば、収束ビームに代えて平行光を照射し、検出面に実像を結像させているため、４次元という高次元を３次元という低次元に次元減縮可能でき、さらに点光源の走査範囲を、次元を含めて任意に設定できるため、実像観察を通じて観察対象物の高分解能かつ高感度な強度像及び微分位相像を走査性良く取得することが可能となる。 According to the present invention, parallel light is emitted instead of the convergent beam to form a real image on the detection surface, so that it is possible to reduce the dimension of four dimensions from the high dimension of three dimensions to the low dimension of three dimensions. Since the scanning range can be arbitrarily set including the dimension, it is possible to acquire the high-resolution and high-sensitivity intensity image and differential phase image of the observation object with good scannability through real image observation.
本発明の原理である４次元相反定理の概説図である。It is a schematic diagram of the four-dimensional reciprocity theorem that is the principle of the present invention. 検出面と光源面の明視野円盤の電子線偏向に伴う重心移動の概説図である。It is a schematic diagram of the movement of the center of gravity due to the electron beam deflection of the bright field disk of the detection surface and the light source surface. 感度向上のための4D-STEMにおける絞り中央遮蔽法と4D-TEMにおける光源部分域走査法の概説図である。FIG. 3 is a schematic diagram of a diaphragm central shielding method in 4D-STEM for improving sensitivity and a light source sub-region scanning method in 4D-TEM. 次元縮減のための１次元光源走査3D-TEM法の概説図である。Ａ，斜光照明走査を行うための偏向コイルを備えたTEM装置の概説図である。Ｂ．１次元走査3D-TEMで用いられる横長スリット絞り（矢印が光源走査方向）。It is a schematic diagram of the one-dimensional light source scanning 3D-TEM method for dimension reduction. A, It is a schematic diagram of a TEM apparatus provided with a deflection coil for performing oblique illumination scanning. B. Horizontal slit diaphragm used in one-dimensional scanning 3D-TEM (arrow indicates the light source scanning direction). 3D-TEMにおいて電子線偏向がない場合の横長スリット絞りを用いた開口全走査（光源全域1次元走査）法説明図である。FIG. 3 is an explanatory view of a full aperture scanning (one-dimensional scanning of the entire light source) using a laterally long slit diaphragm in the absence of electron beam deflection in 3D-TEM. 3D-TEMにおいて電子線偏向がある場合の横長スリット絞りを用いた開口全走査（光源全域1次元走査）法説明図である。FIG. 3 is an explanatory diagram of a full aperture scanning (one-dimensional scanning over the entire light source) method using a laterally elongated slit diaphragm when electron beam is deflected in 3D-TEM. 3D-TEMにおいて電子線偏向がない場合の横長スリット絞りを用いた開口端走査（光源部分域1次元走査）法説明図である。FIG. 3 is an explanatory diagram of an aperture edge scanning (one-dimensional scanning of light source partial region) method using a laterally long slit diaphragm when there is no electron beam deflection in 3D-TEM. 3D-TEMにおいて電子線偏向がある場合の横長スリット絞りを用いた開口端走査（光源部分域1次元走査）法説明図である。FIG. 3 is an explanatory diagram of an aperture edge scanning (one-dimensional scanning of light source partial region) method using a laterally long slit diaphragm when electron beam is deflected in 3D-TEM. 開口端走査（光源部分域1次元走査）法のヴァリエーションを示す図であり、（Ａ）両端両側走査法、（Ｂ）片端両側走査法、（Ｃ）両端片側走査法Ｉ、（Ｄ）両端片側走査法II。It is a figure which shows the variation of the aperture edge scanning (light source partial area one-dimensional scanning) method, (A) both ends both sides scanning method, (B) one end both sides scanning method, (C) both ends one side scanning method I, (D) both ends one side Scanning method II. 本発明の第１の実施形態の透過型顕微鏡装置の構成例を示すブロック図である。It is a block diagram which shows the structural example of the transmission type microscope apparatus of 1st Embodiment of this invention. 本発明の第１の実施形態で用いられるデータ処理方法を示すフローチャートである。3 is a flowchart showing a data processing method used in the first embodiment of the present invention.
　以下、本発明を実施するための形態について、添付の図面を参照して、詳細に説明する。なお、本発明は、以下に説明する実施形態に限定されるものではない。また、説明は以下順に行う。
１．従来法における微分位相量の観測原理
２．第１グループの実施形態
：収束ビーム照射の代わりに平行光照射を用い、点光源または絞りの２次元走査を行い、得られた４次元データから微分位相像を演算取得する観察法
３．第２グループの実施形態
：収束ビーム照射の代わりに平行光照射を用い、点光源または絞りの１次元走査を行い、得られた３次元データから微分位相像を演算取得する観察法
４．第３グループの実施形態
：収束ビーム照射の代わりに平行光照射を用い、点光源または絞りにつき走査範囲を絞り開口端近傍に限定して１次元走査を行い、得られた３次元データから微分位相像を演算取得する観察法 Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. The present invention is not limited to the embodiments described below. The description will be given in order below.
1. Observation principle of differential phase amount in the conventional method 2. 1. Embodiment of the first group: An observation method in which parallel light irradiation is used instead of convergent beam irradiation, two-dimensional scanning of a point light source or an aperture is performed, and a differential phase image is calculated and acquired from the obtained four-dimensional data. Second group embodiment: Observation method in which parallel light irradiation is used instead of convergent beam irradiation, one-dimensional scanning of a point light source or a diaphragm is performed, and a differential phase image is calculated and acquired from the obtained three-dimensional data. Third group of embodiments: parallel light irradiation is used instead of convergent beam irradiation, one-dimensional scanning is performed with a point light source or diaphragm limited to a scanning range near the aperture opening end, and differential phase is obtained from the obtained three-dimensional data. Observation method for calculating and acquiring images
（従来法における微分位相量の観測原理）
［収束ビームの偏向と微分位相］
　図１Ａは、前述した収束ビームの偏向を用いた従来の収束ビーム走査回折像観察４次元顕微鏡(4D-STEM)の結像過程の概略を示す図である。なお、４次元顕微鏡における「４次元」は、前述したように、光源面２次元座標と検出面２次元座標を独立に扱う本発明の基本理念の表現である。図１Ａに示す結像光学系は、４つの光学面を備えており、第１光学面が光源面、第２光学面が絞り面、第３光学面が物面、第４光学面が検出面となる。この光学系１００では、第１レンズ１０１の前焦点面にある光源面１１１上の点から射出した光が第１レンズ１０１、絞り１１２、第２レンズ１０２を通過し、収束ビームとして物面１１３上の観察対象物に照射される。 (Observation principle of the differential phase amount in the conventional method)
[Deflection of convergent beam and differential phase]
FIG. 1A is a diagram schematically showing an image forming process of a conventional focused beam scanning diffraction image observation four-dimensional microscope (4D-STEM) using the above-described focused beam deflection. As described above, "four-dimensional" in a four-dimensional microscope is an expression of the basic idea of the present invention in which the two-dimensional coordinates of the light source surface and the two-dimensional coordinates of the detection surface are handled independently. The imaging optical system shown in FIG. 1A includes four optical surfaces, the first optical surface is a light source surface, the second optical surface is a diaphragm surface, the third optical surface is an object surface, and the fourth optical surface is a detection surface. Becomes In this optical system 100, light emitted from a point on the light source surface 111 on the front focal plane of the first lens 101 passes through the first lens 101, the diaphragm 112, and the second lens 102, and is focused on the object plane 113 as a converged beam. The object to be observed is irradiated.
　観察対象物により偏向した収束ビームは、第３レンズ１０３を通過し、後焦点面に設置された検出面１１４上に絞りの像である検出面明視野円盤を形成する。この検出面明視野円盤の移動が微分位相を与え、光源位置を走査すれば微分位相走査像が得られる。光による物の結像過程は、以下見るように４つの光学面をつなぐ３つのレンズ１０１～１０３によるフーリエ変換過程でもある。 The convergent beam deflected by the observation object passes through the third lens 103, and forms a detection surface bright-field disc, which is an image of the diaphragm, on the detection surface 114 installed on the back focal plane. The movement of the detection surface bright field disk gives a differential phase, and a differential phase scanning image is obtained by scanning the light source position. The image formation process by light is also a Fourier transform process by three lenses 101 to 103 connecting four optical surfaces, as will be seen below.
　具体的には、第１レンズ１０１の前焦点面にある光源面１１１上の１点ｒｓから射出された光δ（ｒ）は、第１レンズ１０１を通過する際に逆フーリエ変換(ＦＴ－１)で表される作用を受け、絞りにおいて絞り関数Ｐ（ｓ）で表される作用を受け、第２レンズ１０２を通過する際にフーリエ変換(ＦＴ)で表される作用を受け、プローブ関数ｐ（ｒｓ－ｒ）で表される収束ビームとしてｔ（ｒ）で表される物面１１３に照射される。そして、物で偏向した収束ビームは、第３レンズ１０３を通過する際にフーリエ変換ＦＴで表される作用を受け、後焦点面に置かれた検出面１１４上に下記数式１で表される検出面明視野円盤を形成する。 Specifically, the light δ (r) emitted from one point r s on the light source surface 111 on the front focal plane of the first lens 101 is subjected to an inverse Fourier transform (FT − ) when passing through the first lens 101. Under the action represented by 1 ), the action represented by the aperture function P (s) at the aperture, the action represented by the Fourier transform (FT) when passing through the second lens 102, and the probe function. The convergent beam represented by p(r s −r) is applied to the object surface 113 represented by t(r). Then, the convergent beam deflected by the object is subjected to the action represented by the Fourier transform FT when passing through the third lens 103, and the detection represented by the following formula 1 is performed on the detection surface 114 placed on the back focal plane. Form a bright field disc.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　この検出面明視野円盤の移動が微分位相を与え、光源位置を走査すれば、照射レンズ系により収束する光（ビーム）は観察対象物を走査するので、微分走査像が得られる。このように光（ビーム）による物の結像過程は、４つの光学面をつなぐ３つのレンズによるフーリエ変換過程でもある。ちなみに、絞り関数Ｐ（ｓ）とプローブ関数ｐ（ｒ）はフーリエ変換でむすばれている。 If the movement of the detection surface bright-field disk gives a differential phase and the light source position is scanned, the light (beam) converged by the irradiation lens system scans the observation object, so that a differential scanning image can be obtained. In this way, the imaging process of an object with light (beam) is also a Fourier transform process by three lenses that connect four optical surfaces. Incidentally, the diaphragm function P(s) and the probe function p(r) are connected by Fourier transform.
　ところで、検出面明視野円盤の移動を定量するのに必要な検出器の大きさは、検出面明視野円盤の移動幅を考慮して、最大移動幅を付加した検出面明視野円盤直径の大きさが下限となる。検出面明視野円盤自体は、絞り面１１２に配置された絞りの像なので、その大きさは絞りの大きさに比例し、空間分解能を良くするため収束ビームを小さく収束すればするほど逆比例関係にある絞りは大きくなり、対応する検出器も大きくなる。一方、微分位相の観測精度は、検出器の空間解像度に比例する。検出器の要求仕様は、このように求めたい画像空間分解能と微分位相観測精度の両者に依存する。 By the way, the size of the detector necessary for quantifying the movement of the detection surface bright field disk is the size of the diameter of the detection surface bright field disk with the maximum movement width added considering the movement width of the detection surface bright field disk. Is the lower limit. Since the detection surface bright-field disk itself is an image of the diaphragm arranged on the diaphragm surface 112, its size is proportional to the size of the diaphragm, and the smaller the convergent beam is converged in order to improve the spatial resolution, the more inversely proportional the relationship is. The aperture at 2 is larger and the corresponding detector is also larger. On the other hand, the observation accuracy of the differential phase is proportional to the spatial resolution of the detector. The required specifications of the detector depend on both the image spatial resolution and the differential phase observation accuracy to be obtained in this way.
　一般に、観察対象物を透過する光波や電子波などの搬送波は、屈折率に依存した位相変化を受ける。屈折率は空間的に分布するので、対応する位相も空間的に分布する。その際、位相と屈折率の関係は下記数式２により表される。 Generally speaking, carriers such as light waves and electron waves that pass through the observation object undergo a phase change depending on the refractive index. Since the refractive index is spatially distributed, the corresponding phase is also spatially distributed. At that time, the relationship between the phase and the refractive index is represented by the following mathematical formula 2.
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　なお、上記数式２において、θ（ｒ）は位相分布、ｎ（ｒ）は屈折率分布、ｌ（ｒ）は厚さの分布、ｒは２次元空間座標、λは搬送波の波長を表す。また、（ｎ（ｒ）－１）の「１」は空気（正確には真空）の屈折率であり、物がある時とない時のその場所の屈折率の変化が、位相変化に対応している。そして、（ｎ（ｒ）－１）ｌ（ｒ）が光学距離に相当する。 In the above equation 2, θ (r) is a phase distribution, n (r) is a refractive index distribution, l (r) is a thickness distribution, r is a two-dimensional spatial coordinate, and λ is a wavelength of a carrier wave. In addition, “1” of (n(r)−1) is the refractive index of air (to be exact, vacuum), and the change in the refractive index at the place with and without an object corresponds to the phase change. ing. Then, (n(r)-1)l(r) corresponds to the optical distance.
　屈折率の分布は、微小範囲で見れば小さなプリズムの分布と近似できる。微小プリズムは、微小部分の屈折率の空間変化であり、上記数式１の関係から、位相の空間変化に対応することになり、結局位相空間分布の勾配（２次元１次微分＝∇θ（ｒ）＝(∂θ（ｒ）／∂x, ∂θ（ｒ）／∂y））に比例する。一方、観察対象物を微小プリズムの集まりと考えた場合、収束光（ビーム）で狭い部分を照明すると、光（ビーム）は微小プリズムにより入射方向とは異なる方向に曲げられる。これが収束光（ビーム）を観察対象物に照射したときに起こる光線（ビーム）偏向の物理過程である。 The distribution of the refractive index can be approximated to that of a small prism when viewed in a minute range. The minute prism is a spatial change in the refractive index of a minute portion, which corresponds to the spatial change in the phase from the relationship of the above-mentioned expression 1, and eventually the gradient of the phase space distribution (two-dimensional first derivative = ∇θ(r ) = (∂θ (r) / ∂x, ∂θ (r) / ∂y)). On the other hand, when the observation target is considered as a group of micro prisms, when a narrow portion is illuminated with convergent light (beam), the light (beam) is bent by the micro prisms in a direction different from the incident direction. This is the physical process of ray (beam) deflection that occurs when the focused light (beam) is applied to the observation object.
　従って、収束ビームの偏向を定量できれば、微分位相量を観測することができる。そして、偏向量の最も簡単な方法は、観察対象物に照射された収束ビームにより、検出面に作出される検出面明視野円盤の偏向に伴う空間移動を定量することである。 Therefore, if the deflection of the convergent beam can be quantified, the differential phase amount can be observed. Then, the simplest method of the deflection amount is to quantify the spatial movement associated with the deflection of the detection surface bright-field disc created on the detection surface by the convergent beam irradiated on the observation object.
［収束ビーム照射とその走査による従来の微分位相像観察法］
　最も簡略化した顕微鏡光学系は、対物レンズと接眼レンズとで構成される２レンズ系であるが、本実施形態の顕微鏡観察方法では、光源面と検出面でそれぞれ独立に定義された光源面２次元座標と検出面２次元座標を用いて表される４次元座標に基づいて４次元的観察を行うため、光源面を明示的に定義する必要がある。そこで、本実施形態の顕微鏡観察方法では、図１Ａで示したような光源面と集光レンズ（コレクターレンズ）を含めた３レンズ系を採用する。 [Conventional differential phase image observation method by focused beam irradiation and its scanning]
The most simplified microscope optical system is a two-lens system composed of an objective lens and an eyepiece lens. In the microscope observation method of this embodiment, the light source surface 2 defined independently of the light source surface and the detection surface is used. Dimensional coordinates and detection surface Since four-dimensional observation is performed based on four-dimensional coordinates expressed using two-dimensional coordinates, it is necessary to explicitly define the light source surface. Therefore, in the microscope observation method of the present embodiment, a three-lens system including a light source surface and a condenser lens (collector lens) as shown in FIG. 1A is adopted.
　３レンズ・４光学面系の扱いをさらに単純化するため、本実施形態の顕微鏡観察方法では、３つのレンズは同じ焦点距離ｆを持ち、レンズと光学面合わせて７つの要素が同じ焦点距離間隔で結合される３レンズ・４光学面・６焦点系とする。ここでは、顕微鏡の拡大機能は考えないので、結像倍率は１倍として扱う。また、本実施形態においては、レンズ作用を、物理的に扱うときは光の収束効果、数理的に扱うときはフーリエ変換作用として扱う。 In order to further simplify the handling of the 3-lens and 4-optical surface system, in the microscope observation method of this embodiment, the three lenses have the same focal length f, and the seven elements having the same optical distance as the lenses have the same focal length interval. 3 lenses, 4 optical surfaces, and 6 focal point system combined by. Since the enlargement function of the microscope is not considered here, the imaging magnification is treated as 1. Further, in the present embodiment, the lens action is treated as a light converging effect when physically treated, and as a Fourier transform action when treated mathematically.
　３レンズ・４光学面系の光学系において、収束ビームを走査しながら照射する従来の微分位相像観察法を図示したのが図１Ａである。ここで、検出面明視野円盤を現出させる光場を、光源面の実空間座標ｒｓと検出面の周波数空間座標ｓｄを用いてＵSTEM（ｒｓ，ｓｄ）と表記する。４次元関数の変数である２つの２次元座標の順序は、光源面座標を最初に、検出面座標を２番目に置いた。ＵSTEM（ｒｓ，ｓｄ）は、個別ｒｓをパラメーターとしてｓｄ座標検出面に展開する回折像2次元関数であり、物面に共役な光源面上の光源点ｒｓを2次元走査して初めて4次元となる。なお微分位相は、収束ビームの偏向に伴う検出面明視野円盤の移動で表されるので、具体的な計算は、光場により形成されるｓｄ座標強度像（２乗検出像）としての検出面明視野円盤関数、｜ＵSTEM（ｒｓ，ｓｄ）｜２の重心計算を用いて行われる。これを、検出面におけるｓｄ重心と呼ぶ。 FIG. 1A illustrates a conventional differential phase image observation method in which a convergent beam is irradiated while being scanned in an optical system of a 3-lens / 4-optical surface system. Here, the optical field for revealing the detection MenAkira viewing disc, denoted U STEM (r s, s d ) and using the frequency space coordinates s d of the real space coordinate r s and the detection surface of the light source plane. Regarding the order of the two two-dimensional coordinates which are variables of the four-dimensional function, the light source plane coordinate was placed first and the detection plane coordinate was placed second. U STEM (r s, s d ) is a diffraction image two-dimensional function to expand the s d coordinate detection plane individual r s as a parameter, the object plane 2 dimensionally scanning the source point r s on conjugate light plane Only then will it be four-dimensional. Since the differential phase is represented by the movement of the bright-field disc on the detection surface due to the deflection of the convergent beam, the specific calculation is the detection as an s d coordinate intensity image (square detection image) formed by the optical field. MenAkira field disc function, | U STEM (r s, s d) | is performed using the second center of gravity calculation. This is called the s d centroid on the detection surface.
　一般に、顕微鏡においては、観察対象物に収束ビームを照射し、その収束ビームを２次元走査し、検出面上に置いた広角検出器で回折像の全強度を取得することで、操作的に２次元像を取得する走査法と、観察対象物に平行光を照射し、走査なしの１回の照射により生成される２次元実像を検出面上に置いた2次元検出器で取得する平行光照射法がある。前述したように、収束ビーム照射の場合、照明光は観察対象物の狭い領域に当たるので、局所的に偏向度合いを特定することができる。一方、平行光照射の場合、照明光が観察対象物の広い領域に照射されて四方八方に偏向されるため、観察対象物上の局所での偏向度合いを特定し、場所依存的には微分位相を抽出することはできないと考えられてきた。 Generally, in a microscope, an object to be observed is irradiated with a convergent beam, the convergent beam is two-dimensionally scanned, and the total intensity of the diffraction image is acquired by a wide-angle detector placed on a detection surface. A scanning method for acquiring a dimensional image and parallel light irradiation for irradiating an observation object with parallel light and acquiring a two-dimensional real image generated by one irradiation without scanning with a two-dimensional detector placed on the detection surface. There is a law. As described above, in the case of the convergent beam irradiation, the illumination light strikes a narrow area of the observation target, so that the degree of deflection can be specified locally. On the other hand, in the case of parallel light irradiation, the illumination light is irradiated to a wide area of the observation object and is deflected in all directions, so the degree of local deflection on the observation object is specified, and the differential phase is dependent on the location. It has been considered impossible to extract.
(平行光照射による４次元透過型顕微鏡(4D-TEM)法による微分位相量観測原理)
　先ず、本発明の基本である平行光照射による４次元透過型顕微鏡(4D-TEM)法による微分位相両の観測原理について説明する。本発明者は、光の逆進の性質を用い、かつ、通常の２次元的観察ではなく、４次元的観察を行えば、平行光照射法でも微分位相の観察が可能であることを見出し、本発明に至った。図１Ａは収束ビームを適用した従来型の走査型４次元法(4D-STEM)の結像図であり、図1Ｂは多数の点光源から射出される平行照明光を模式化した本発明の平行光照射型４次元法(4D-TEM)の結像図である。ただし、多数の点光源からの同時照射実験はないので、図1Ｂは異なる個別的点光源実験の重ね合わせを概念的に示したものである。図1Ａと図1Ｂは鏡映対象であり、電子顕微鏡ですでに見出されている収束照明光を用いる走査透過型電子顕微鏡（STEM）と平行照明光を用いる透過型電子顕微鏡（TEM）の間に成立するTEM-STEM相反定理の４次元法への拡張であり、以後4次元相反定理と呼ぶ。 (Principle of differential phase amount observation by four-dimensional transmission microscope (4D-TEM) method with parallel light irradiation)
First, the principle of observation of both differential phases by the four-dimensional transmission microscope (4D-TEM) method by parallel light irradiation, which is the basis of the present invention, will be described. The present inventor has found that it is possible to observe the differential phase even by the parallel light irradiation method by using the reverse property of light and performing four-dimensional observation instead of normal two-dimensional observation. The present invention was reached. FIG. 1A is an image diagram of a conventional scanning four-dimensional method (4D-STEM) using a convergent beam, and FIG. 1B is a schematic view of parallel illumination light emitted from a large number of point light sources according to the present invention. It is an image formation figure of a light irradiation type four-dimensional method (4D-TEM). However, since there are no simultaneous irradiation experiments from a large number of point light sources, FIG. 1B conceptually shows the superposition of different individual point light source experiments. 1A and 1B are the objects to be mirrored, which are between the scanning transmission electron microscope (STEM) using the convergent illumination light and the transmission electron microscope (TEM) using the parallel illumination light, which have already been found in the electron microscope. Is an extension of the TEM-STEM reciprocity theorem to a four-dimensional method, and is called the four-dimensional reciprocity theorem.
　収束ビームを適用した走査型４次元法では、光源面１１１上の１つの光源点ｒｓから光線束が射出される。図１Ａに示すように、その様子は例えば代表的な３つの光線で表現される。そして、各光線は、レンズ１０１及びレンズ１０２により物面１１３上の１点に収束され、観察対象物により散乱された後、レンズ１０３により回折されて、検出面１１４の２次元座標上に広がる回折像、明視野円盤として観察される。（明視野円盤は図２に示されている。）図１Ａに示す実線は光線が観察対象物によって局所的に偏向する様子を示し、波線は観察対象物がない時に直進する様子を示している。即ち、図１Ａには、観察対象物による収束ビームの偏向に伴い、検出面１１４上で検出面明視野円盤が移動する様子が明示されている。この移動量が微分位相量に対応する。 In applying the converged beam scanning 4D method, light rays are emitted from a single source point r s on the light source surface 111. As shown in FIG. 1A, the state is represented by, for example, three typical light rays. Then, each light beam is converged by the lens 101 and the lens 102 to one point on the object surface 113, scattered by the observation object, diffracted by the lens 103, and spread on the two-dimensional coordinates of the detection surface 114. The image is observed as a brightfield disc. (The bright-field disk is shown in FIG. 2.) The solid line shown in FIG. 1A shows how the light beam is locally deflected by the observation object, and the wavy line shows how it goes straight when there is no observation object. .. That is, FIG. 1A clearly shows that the detection surface bright-field disk moves on the detection surface 114 as the convergent beam is deflected by the observation object. This amount of movement corresponds to the amount of differential phase.
　光の逆進を利用し、図１Ａに示す結像図を鏡映反転しても光線図は成立する。図１Ｂは、図１Ａの光逆進光線図であり、光源面の多数の点からそれぞれ光軸に対して傾きをもった平行照明光が観察対象物に照射される構成の図である。図１Ｂは、図１Ａの光源面と検出面とを置換した上で、左右逆転させた結像図であり、検出面２４上の１つの像点に集光する光線が、光源面２１上の３つの異なる点光源から射出される様子を示している。この場合の光線図も、物面２２上における観察対象物の有無で異なり、その様子は図１Ｂでそれぞれ実線と破線で示されている。これは、収束ビーム照射における検出面１１４上の明視野円盤の移動に対応し、平行光照射における検出面２４上の１点に収束する多数の点光源からなる光源面の領域（以下、4D-STEMの明視野円盤に対応し、光源面明視野円盤と呼ぶ。）の移動を表し、この移動を定量できれば微分位相量が観察対象物上の各点で求まる。なお図２には4D-STEM法の検出面明視野円盤と対応する4D-TEM法の光源面明視野円盤の両方について、電子線偏向に伴う移動の様子が4次元相反的に描かれている。 The ray diagram can be established even if the image formation diagram shown in FIG. 1A is mirror-inverted by utilizing the backward movement of light. FIG. 1B is a reverse light ray diagram of FIG. 1A, and is a diagram of a configuration in which parallel illumination light, each of which has an inclination with respect to the optical axis, is irradiated onto an observation object from a large number of points on the light source surface. FIG. 1B is an image diagram in which the light source surface and the detection surface in FIG. 1A are replaced with each other and then the images are reversed to the left and right. It is shown that light is emitted from three different point light sources. The ray diagram in this case also differs depending on the presence or absence of the observation object on the object surface 22, and the state is shown by the solid line and the broken line in FIG. 1B. This corresponds to the movement of the bright field disk on the detection surface 114 in the convergent beam irradiation, and the area of the light source surface (hereinafter, 4D-) which is composed of a large number of point light sources that converge to one point on the detection surface 24 in the parallel light irradiation. It corresponds to the bright-field disc of STEM and is called the bright-field disc of the light source surface.) If this movement can be quantified, the differential phase amount can be obtained at each point on the observation object. Note that Fig. 2 shows the movement of both the detection surface bright-field disk of the 4D-STEM method and the corresponding light source surface bright-field disk of the 4D-TEM method due to electron beam deflection in a four-dimensional reciprocity. ..
　光の逆進で重要なのは、全ての光学面が入れ代わることであり、例えば光源面と検出面が入れ代わることである。この場合、光学面の物理的意味は変わるが、各光学面に付与された座標系の数理的性質、即ち、実空間座標系か周波数空間座標系かの性質は保持される。その結果、検出面(detection plane）の周波数空間座標ｓｄはｓｓに、光源面(source plane）の実空間座標ｒｓはｒｄに、それぞれ変更される。（下つきのdおよびsはそれぞれdetection plane、source planeを意味している。）前述した収束ビーム照射４次元法の検出面光場の標記法ＵＳＴＥＭ（ｒｓ，ｓｄ）に従うと、平行光（parallel light）を用いる平行光照射４次元法の検出面光場はＵＴＥＭ（ｓｓ，ｒｄ）と表記される。ＵＴＥＭ（ｓｓ，ｒｄ）は、個別ｓｓをパラメーターとしてｒｄ座標検出面に展開する実像2次元関数であり、絞り面に共役な光源面上の光源点ｓｓを2次元走査して初めて4次元となる。なお微分位相の計算は、個別ｒｄに対応して計算的に生成されるｓｓ座標強度像（２乗検出像）｜ＵＴＥＭ（ｓｓ，ｒｄ）｜２に対して実行される。 What is important in the backward movement of light is that all optical surfaces are interchanged, for example, the light source surface and the detection surface are interchanged. In this case, although the physical meaning of the optical surface changes, the mathematical property of the coordinate system given to each optical surface, that is, the property of the real space coordinate system or the frequency space coordinate system is maintained. As a result, the frequency space coordinates s d are s s of the detection surface (detection plane), the real space coordinates r s of the light source surface (source plane) to r d, are changed respectively. (Which means each d and s the subscript detection plane, source plane.) The title method U STEM (r s, s d) of the detection surface optical field of the above-mentioned convergent beam irradiation 4-dimensional method According to parallel light (parallel light) detection surface optical field of the parallel light irradiation 4-dimensional method using is denoted U TEM (s s, r d ) and. U TEM (s s, r d ) is a real two-dimensional function to expand the r d coordinate detection plane individual s s as a parameter, the source point s s on conjugate light source plane is scanned two-dimensionally on the aperture plane For the first time in 4 dimensions. Incidentally calculation of the differential phase, the individual r s s coordinates intensity image are computationally generated corresponding to d (2 square detection image) | is performed for 2 | U TEM (s s, r d).
　平行光照射４次元法の場合、収束ビーム照射４次元法とは異なり、電子線の偏向は、個別ｒｄに対応して仮想的に観測される光源面明視野円盤の移動として現れるので、微分位相量は、ｒｄを固定してｓｓ座標で2次元展開される光源面明視野円盤関数｜ＵＴＥＭ（ｓｓ，ｒｄ）｜２の重心計算を用いて行われる。これを光源面におけるｓｓ重心と呼ぶ。 If parallel light irradiation four-dimensional method, unlike the convergent beam irradiation four-dimensional method, the deflection of the electron beam, so appears as the movement of the light source MenAkira field disc observed virtually corresponds to the individual r d, the derivative phase amount, the light source MenAkira field disc function is expanded in two dimensions s s coordinates securing the r d | U TEM (s s , r d) | is performed using the second center of gravity calculation. This is called the s s center of gravity on the light source surface.
　通常のＴＥＭの平行光照射法では、点光源が光軸上に置かれるが、4D-STEMの平行光照射法では、収束ビーム照射４次元法と同様に、光軸から離れた点から発せられる電子ビームを２次元走査する。 In the normal TEM parallel light irradiation method, the point light source is placed on the optical axis, but in the 4D-STEM parallel light irradiation method, it is emitted from a point away from the optical axis, as in the convergent beam irradiation four-dimensional method. The electron beam is two-dimensionally scanned.
　この場合、光軸から離れた点から観察物対象物を照明すること、即ち、通常の光軸に平行な光（ビーム）の照射に代えて、光軸に対して傾きをもつ光（ビーム）を照射すること（以下、「斜光照明」という。）により、検出面で観察される実像を記録する。そして、光源点を２次元的に走査し、その都度観察像を、例えば顕微鏡装置に設けられたデータ記憶部に蓄積する。こうしてデータは、光源面の２次元座標と実像検出面の２次元座標の合成である４次元座標上に展開され4次元データとなる。 In this case, the object to be observed is illuminated from a point distant from the optical axis, that is, light (beam) having an inclination with respect to the optical axis is used instead of irradiation of light (beam) parallel to the normal optical axis. By irradiating (hereinafter, referred to as “oblique illumination”), a real image observed on the detection surface is recorded. Then, the light source point is two-dimensionally scanned, and each time the observation image is accumulated in the data storage unit provided in the microscope apparatus. In this way, the data is developed into four-dimensional data on the four-dimensional coordinate which is a combination of the two-dimensional coordinate of the light source surface and the two-dimensional coordinate of the real image detecting surface.
　３レンズ・４光学面の７要素で構成される光学系を用いて、収束光（ビーム）照射と平行光（ビーム）照射を比較すると、図３Ａに示すように、収束光（ビーム）照射では、光源面１１１－検出面１１４結像過程の数理はＦＴ－１、ＦＴ及びＦＴの順となる。一方、図１Ｂに示すように、光の逆進に対応し、平行光（ビーム）照射では、光源面２１－検出面２４結像過程の数理はＦＴ、ＦＴ及びＦＴ－１の順となる。 Comparing convergent light (beam) irradiation and parallel light (beam) irradiation using an optical system consisting of 7 elements of 3 lenses and 4 optical surfaces, as shown in FIG. 3A, converged light (beam) irradiation The math of the image formation process of the light source surface 111-the detection surface 114 is in the order of FT -1 , FT and FT. On the other hand, as shown in FIG. 1B, in the case of parallel light (beam) irradiation, the mathematical process of the image formation process of the light source surface 21-detection surface 24 is FT, FT, and FT −1 in the order of parallel light (beam) irradiation.
　そして、収束光（ビーム）照射で得られる４次元像は、下記数式３で表される照射光のプローブ関数ｐ（ｒ）と、下記数式４で表される対象物透過関数ｔ（ｒ）を用いて、下記数式５で表される。なお、プローブ関数ｐ（ｒ）と対象物透過関数ｔ（ｒ）は、共に複素数である。 Then, the four-dimensional image obtained by the convergent light (beam) irradiation has a probe function p(r) of the irradiation light represented by the following formula 3 and an object transmission function t(r) represented by the following formula 4. It is expressed by the following Equation 5. The probe function p (r) and the object transmission function t (r) are both complex numbers.
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000005
　一方、平行光照射で得られる４次元像は、下記数式６で表される。 On the other hand, the four-dimensional image obtained by parallel light irradiation is represented by the following mathematical formula 6.
Figure JPOXMLDOC01-appb-M000006
Figure JPOXMLDOC01-appb-M000006
　ここで、上記数式６における絞り関数Ｐ（ｓ）は下記数式７で表され、照射光プローブ関数とフーリエ変換ＦＴで結ばれる。また、Ｔ（ｓ）は、下記数式８で表され,対象物透過関数とフーリエ変換で結ばれる。なお、Ｐ（ｓ）とＴ（ｓ）は、共に複素数である。 Here, the diaphragm function P(s) in the above formula 6 is expressed by the following formula 7, and is connected to the irradiation light probe function by the Fourier transform FT. Further, T (s) is expressed by the following mathematical formula 8 and is connected to the object transmission function by the Fourier transform. Both P(s) and T(s) are complex numbers.
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000008
Figure JPOXMLDOC01-appb-M000008
　収束光（ビーム）照射による４次元法と平行光（ビーム）照射による４次元法での微分位相量は、上記数式２～８を用いて、それぞれ数式９及び数式１０で表される。 The differential phase amounts in the four-dimensional method by the convergent light (beam) irradiation and the four-dimensional method by the parallel light (beam) irradiation are expressed by the mathematical expressions 9 and 10 using the above mathematical expressions 2 to 8, respectively.
Figure JPOXMLDOC01-appb-M000009
Figure JPOXMLDOC01-appb-M000009
Figure JPOXMLDOC01-appb-M000010
Figure JPOXMLDOC01-appb-M000010
　上記数式９及び数式１０において、分子は１次モーメントを表し、分母は信号強度に対応する面積分を表す。また、割り算は重心に対応し、こうした演算を重心積分という。 In Equations 9 and 10, the numerator represents the first moment and the denominator represents the area corresponding to the signal strength. Further, division corresponds to the center of gravity, and such calculation is called center of gravity integration.
　ｓｄ重心に対する上記数式９は、透過係数の位相成分の微分∇θｔ（ｒ）とプローブ関数の位相成分の微分∇θｐ（ｒ）の双方を含んでいる。微分符号などは若干異なるが、これは、Waddellらにより世界で最初に導出された数式と同じである（非特許文献１参照）。一方、上記数式１０に示すｓｓ重心の表式は、本発明者が発見したものであり、本出願が世界初出である。そして、上記数式９及び数式１０を比較すると、光源面における実座標ｒｓを変数とするか、検出面における実座標ｒｄを変数とするかの違いを除けば、透過係数とプローブ関数の位相成分の微分表現が同じ表式を与えていることがわかる。 The above equation 9 for the s d centroid includes both the derivative ∇θ t (r) of the phase component of the transmission coefficient and the derivative ∇θ p (r) of the phase component of the probe function. Although the differential code and the like are slightly different, this is the same as the mathematical formula first derived in the world by Waddell et al. (see Non-Patent Document 1). On the other hand, the expression of the s s center of gravity shown in the above equation 10 was discovered by the present inventor, and this application is the first in the world. When comparing the above equation 9 and equation 10, or the actual coordinates r s in the light source plane is variable, except of differences as a variable actual coordinates r d in the detection plane, the transmission coefficient and the probe function of phase It can be seen that the differential representation of the components gives the same expression.
　この結果は、図３Ａ，図３Ｂに示す２つの４次元法の光場が光の逆進から考え等価であることを解析的に示していると共に、Waddellらにより提案された収束ビームの観察対象物上走査４次元法が、平行光照射を用いた光源面上点光源走査４次元法に代替できることを意味している。ここで、プローブ関数ｐ（ｒ）について考えると、顕微鏡のプローブ関数は、ほとんどのケースで中心対称なので、位相微分∇θｐ（０）は０となり、ｓｄ重心及びｓｓ重心は、共にｔ（ｒ）の位相成分θｔ（ｒ）の微分のみを用いて表される。ただし、本発明の平行光照射４次元顕微鏡観察方法で用いられる微分は、２次元１次微分、即ち下記数式１１に示す勾配を意味し、∇θｔ（ｒ）は下記数式１２で表される。 This result analytically shows that the optical fields of the two four-dimensional methods shown in FIGS. 3A and 3B are equivalent because they are considered from the backward direction of light, and the observation target of the convergent beam proposed by Waddell et al. This means that the four-dimensional scanning method on the object can be replaced with the four-dimensional scanning method of point light source on the light source surface using parallel light irradiation. Here, considering the probe function p(r), since the probe function of the microscope is centrally symmetric in most cases, the phase differential ∇θ p (0) is 0, and the s d centroid and the s s centroid are both t. It is expressed using only the derivative of the phase component θ t (r) of (r). However, the differential used in the parallel light irradiation four-dimensional microscope observation method of the present invention means a two-dimensional first-order differential, that is, the gradient shown in the following formula 11, and ∇θ t (r) is represented by the following formula 12. ..
Figure JPOXMLDOC01-appb-M000011
Figure JPOXMLDOC01-appb-M000011
Figure JPOXMLDOC01-appb-M000012
Figure JPOXMLDOC01-appb-M000012
　そして、プローブ関数ｐ（ｒ）がδ関数的に絞った収束ビームであれば、上記数式９及び数式１０は、それぞれ下記数式１３及び数式１４に帰着される。 Then, if the probe function p(r) is a convergent beam narrowed down by a δ function, the above equations 9 and 10 are reduced to the following equations 13 and 14, respectively.
Figure JPOXMLDOC01-appb-M000013
Figure JPOXMLDOC01-appb-M000013
Figure JPOXMLDOC01-appb-M000014
Figure JPOXMLDOC01-appb-M000014
　以上から、収束光照射による４次元顕微鏡データ及び平行光照射による４次元顕微鏡データの両方について数式９及び数式１０に示すｓ重心計算を行えば、観察対象物の微分位相が求まることが分かる。実験で得られた∇θ(r)（２次元）からθ（r）（１次元）を得るには、勾配からポテンシャルを求める定法に従い、下記数式１５に示す線積分操作を行えばよい。 From the above, it can be seen that the differential phase of the observation object can be obtained by performing the s center of gravity calculation shown in Equations 9 and 10 for both the four-dimensional microscope data by the focused light irradiation and the four-dimensional microscope data by the parallel light irradiation. In order to obtain θ (r) (1D) from ∇θ (r) (2D) obtained in the experiment, the line integral operation shown in Equation 15 below may be performed according to the formula for obtaining the potential from the gradient.
Figure JPOXMLDOC01-appb-M000015
Figure JPOXMLDOC01-appb-M000015
　上記数式１５において、積分路Ｃは、例えばある起点ｒｉ＝（ｘｉ，ｙｉ）からｘ軸に平行な直線（ｘ，ｙｉ）、その直線端点（ｘｆ，ｙｉ）でｙ軸に平行な直線（ｘｆ，ｙ）で、終点が求めたいｒｆ＝（ｘｆ，ｙｆ）点となるような道筋である。数式１５はまた位相を回復するのに直交する２つの微分位相成分があれば充分であることを示している。 In the above equation 15, the integration path C is, for example, a straight line (x, y i ) parallel to the x-axis from a certain starting point r i = (x i , y i ), and the y-axis at the linear end point (x f , y i ). Is a straight line (x f , y) parallel to, and the end point is the path to be the desired r f =(x f , y f ) point. Equation 15 also shows that it is sufficient to have two orthogonal differential phase components to restore the phase.
　また、上記数式９及び数式１０で周波数空間の重心（ｓ重心）を計算するとき、下記数式１６に示すｔ（ｒ）の強度情報は、分母に現れる光場２乗検出の全積分値として得られているので、観察対象物の光学情報、位相と強度の両方が得られたことになる。 Further, when the center of gravity (s center of gravity) of the frequency space is calculated by the above formulas 9 and 10, the intensity information of t (r) shown in the following formula 16 is obtained as the total integral value of the light field squared detection appearing in the denominator. Therefore, both the optical information, phase and intensity of the observation object have been obtained.
Figure JPOXMLDOC01-appb-M000016
Figure JPOXMLDOC01-appb-M000016
[高感度化の方法]
　4D-STEM法の難点である有機物不適用に関して4D-TEMは以下に述べる高感度化を実現できる。まず図２を見ると明視野円盤の移動が小さい場合、その変化は移動方向に直交する絞り周辺部の狭い領域で生じていることが分かる。これは重心移動を与える信号が絞り周辺部の狭い領域に集中していることを意味している。事実4D-STEMの観測データにおいて重心移動を与える信号が不均等であり絞り、周辺部分の積分走査で高感度の微分位相が求められるとの報告がある（非特許文献１３）。これを4D-TEMに応用した場合、光源の一様均等走査の代わりに、絞り周辺部の部分走査を行うことで高感度の新規４次元観察法が可能性となることに思いいたる。 [Method of increasing sensitivity]
Regarding the non-application of organic substances, which is a difficulty of 4D-STEM method, 4D-TEM can realize the high sensitivity described below. First, referring to FIG. 2, when the movement of the bright-field disk is small, it can be seen that the change occurs in a narrow area around the diaphragm orthogonal to the moving direction. This means that the signals that give the movement of the center of gravity are concentrated in a narrow area around the aperture. In fact, it has been reported that the signal giving the movement of the center of gravity is uneven in the observation data of 4D-STEM, the aperture is narrowed down, and a highly sensitive differential phase is required by the integral scanning of the peripheral portion (Non-Patent Document 13). When this is applied to 4D-TEM, we think that a new high-sensitivity four-dimensional observation method will become possible by performing partial scanning of the peripheral area of the diaphragm instead of uniform scanning of the light source.
　具体的には図３に示すように、重心移動に寄与しない明視野円盤の中央部分をデータから除く観測を行うため、4D-STEMでは絞りの中央に大きな遮蔽板を、4D-TEMでは光源走査を絞り周辺部分に限定する部分走査、具体的には開口端走査行う。この方法は見る必要のない部分の電子線資源を、信号を担う絞り周辺部対応の信号箇所に集中できるので信号対雑音比が増大し感度が向上する。ただし同じ電子線不均等照射でも4D-STEMと4D-TEMには差があり、4D-STEMでは絞りを遮蔽するため分解能が犠牲になる。他方4D-TEMでは絞りに対し遮蔽等の形状変化を与えず、光源走査範囲の制御のみで実現されるので分解能は自由に設定できる。光源部分走査の4D-TEM法は顕微鏡における根源的問題、分解能と感度の相克を始めて克服した。 Specifically, as shown in Fig. 3, in order to perform observations that exclude the central part of the bright-field disk that does not contribute to the movement of the center of gravity from the data, 4D-STEM uses a large shielding plate in the center of the aperture, and 4D-TEM scans the light source. Is limited to the peripheral portion of the diaphragm, specifically, the aperture end scanning is performed. According to this method, the electron beam resources of the portion that does not need to be seen can be concentrated in the signal portion corresponding to the peripheral portion of the diaphragm that carries the signal, so that the signal-to-noise ratio is increased and the sensitivity is improved. However, even with the same electron beam uneven irradiation, there is a difference between 4D-STEM and 4D-TEM, and 4D-STEM sacrifices resolution because it blocks the diaphragm. On the other hand, in 4D-TEM, the resolution can be set freely because it is realized only by controlling the light source scanning range without giving shape changes such as shielding to the diaphragm. The 4D-TEM method of partial scanning of the light source overcomes the fundamental problem in the microscope, the conflict of resolution and sensitivity for the first time.
[次元縮減の方法]
　４次元法の他の難点である高次元性に関しては、4D-TEMで特徴的な次元縮減法がある。図４Ａに示す斜光照明可能なTEMに付きその手法を説明する。図１～３から４次元法の真髄は偏向電子線の絞りによる蹴られにあることが分かる。逆に絞りがなければ明視野円盤は無限に広がり重心位置を特定できない。この原理を生かせば１方向に無限に開いた絞りすなわち横長スリット（図４Ｂ）を用いれば、電子線の蹴られは長手方向に直交したスリット端のみで起こるので、その方向の微分位相が得られることになる。これは可視光顕微鏡で多用される微分干渉コントラスト（DIC）顕微鏡と同じ画像を与える。その１方向の微分なので走査もスリット長手方向に直交した１次元走査となり、４次元データは３次元データへと次元縮減できる。これは観測の効率化をも実現させる。感度向上で述べた部分走査法についても次元縮減が適用でき走査範囲低減と合わせ観測時間はさらに縮小される。 [Method of dimension reduction]
As for the high dimensional property which is another difficulty of the 4-dimensional method, there is a characteristic dimensional reduction method in 4D-TEM. The method will be described for the TEM capable of oblique illumination shown in FIG. 4A. From FIGS. 1 to 3, it can be seen that the essence of the four-dimensional method lies in the kicking by the diaphragm of the polarized electron beam. On the contrary, if there is no diaphragm, the bright-field disk will expand infinitely and the center of gravity cannot be specified. If this principle is utilized, if a diaphragm that is infinitely open in one direction, that is, a horizontally long slit (FIG. 4B) is used, the kicking of the electron beam occurs only at the slit end orthogonal to the longitudinal direction, so that the differential phase in that direction can be obtained. It will be. This gives the same image as the differential interference contrast (DIC) microscope often used in visible light microscopes. Since the differentiation is in one direction, the scanning is also one-dimensional scanning orthogonal to the slit longitudinal direction, and the four-dimensional data can be reduced to three-dimensional data. This also makes the observation more efficient. The dimension reduction can also be applied to the partial scanning method described in the sensitivity improvement, and the observation time can be further shortened by reducing the scanning range.
[感度向上と次元縮減を組み合わせる方法]
　次に感度向上と次元縮減を組み合わせた実験法につき説明する。図５はこの方法の基本形を模式的に示しており、横長スリットに直交する方向に光源の１次元走査を行う。走査領域はスリットの幅を越え観察対象の電子線偏向の最大値までをカバーしスリットの全域を走査するように設定される。図にはスリットに対応するスリット型光源面明視野円盤（術語として円盤という表現を語用するが、１次元走査の場合には、実際に観測されるのは、長手方向に直交する光源走査線上の１次元信号なので明視野円盤ではなく明視野杖となる。）がスリットにピッタリ重なるように光源面に投影して描かれており、光源走査点はスリット中央を横切る点列として表示されている。図５では偏向がないのでこの点列のｙ方向重心は原点と同じである。 [Method to combine sensitivity improvement and dimension reduction]
Next, an experimental method combining sensitivity improvement and dimension reduction will be described. FIG. 5 schematically shows the basic form of this method, in which one-dimensional scanning of the light source is performed in the direction orthogonal to the lateral slit. The scanning area is set so as to cover the width of the slit up to the maximum value of the electron beam deflection of the observation target and scan the entire area of the slit. In the figure, the slit type light source surface bright field disc corresponding to the slit (the expression disc is used as a terminology, but in the case of one-dimensional scanning, what is actually observed is on the light source scanning line orthogonal to the longitudinal direction. Is a one-dimensional signal of the above, so it is not a bright-field disk but a bright-field cane.) is projected and drawn on the light source surface so as to exactly overlap with the slit, and the light source scanning point is displayed as a row of points across the slit center. .. In FIG. 5, since there is no deflection, the y-direction center of gravity of this point sequence is the same as the origin.
　図６は電子線が偏向した場合においてスリット型光源面明視野円盤、スリット、光源点列を模式的に示しており、図５と同じように横長スリットに直交する方向に光源の１次元走査を行う。走査領域はスリットの幅を越え観察対象の電子線偏向の最大値までをカバーしスリットの全域を走査するように設定される。このとき偏向がｙ軸の負方向の生じ、点列のｙ方向重心は偏向に比例し原点から下方に移動する。これで負の微分位相が得られる。 FIG. 6 schematically shows a slit-type light source surface clear-field disk, slits, and a sequence of light source points when the electron beam is deflected, and one-dimensional scanning of the light source is performed in a direction orthogonal to the horizontally long slit as in FIG. To do. The scanning area is set so as to cover the width of the slit up to the maximum value of the electron beam deflection of the observation target and scan the entire area of the slit. At this time, the deflection occurs in the negative direction of the y-axis, and the y-direction center of gravity of the point sequence moves downward from the origin in proportion to the deflection. This gives a negative differential phase.
　図７は、スリット型光源面明視野円盤とスリットの関係は図５と同じだが、光源走査範囲がスリット上下端近傍に限定されている。スリット中央部分の信号はカットされるが、電子線偏向がなければ図５と同じように、この部分走査で得られるデータのｙ方向重心は原点と同じであり移動はない。 In Fig. 7, the relationship between the slit type light source surface bright field disk and the slit is the same as in Fig. 5, but the light source scanning range is limited to the vicinity of the upper and lower ends of the slit. Although the signal at the central portion of the slit is cut, if there is no electron beam deflection, the y-direction center of gravity of the data obtained by this partial scanning is the same as the origin and does not move, as in FIG.
　図８は、スリット型光源面明視野円盤とスリットの関係は図６と同じだが、光源走査範囲がスリット上下端近傍に限定されている。図６と同じようにこの部分走査で得られるデータのｙ方向重心は下方に移動する。このとき観測される重心移動量は、図6に示す全域走査法で観測される重心移動量と同じである。にもかかわらず、図６で示す全域走査の総電子線量を狭い走査範囲に振り向けるので、各走査点での電子線量が増え相対的に電子線観測特有のショットノイズが減少し信号対雑音比は向上する。すなわち感度が向上する。
　以下従前の原理発明に従って構成される種々の実施形態について記述する。 In FIG. 8, the relationship between the slit type light source surface bright field disk and the slit is the same as in FIG. 6, but the light source scanning range is limited to the vicinity of the upper and lower ends of the slit. As in FIG. 6, the y-direction center of gravity of the data obtained by this partial scan moves downward. The amount of movement of the center of gravity observed at this time is the same as the amount of movement of the center of gravity observed by the whole-area scanning method shown in FIG. Nevertheless, since the total electron dose of the whole area scanning shown in FIG. 6 is directed to a narrow scanning range, the electron dose at each scanning point increases and the shot noise peculiar to electron beam observation decreases relatively, and the signal-to-noise ratio. Will improve. That is, the sensitivity is improved.
Hereinafter, various embodiments configured according to the conventional principle invention will be described.
（第１の実施形態）
第１の実施形態は、従前説明した４次元透過型電子顕微鏡原理を忠実に実行するものであり、点光源から射出された光を観察対象物に任意の角度で斜め方向から平行照射する照明工程と、前記観察対象物で偏向され、前記点光源と共役な位置に配置された絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、前記点光源を２次元走査しながら前記照明工程と前記結像工程を行い、光源面２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、前記４次元データを、前記光源面２次元座標における重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する。 (First embodiment)
The first embodiment faithfully executes the four-dimensional transmission electron microscope principle described above, and an illumination step of irradiating an object to be observed with light emitted from a point light source in parallel from an oblique direction at an arbitrary angle. An imaging step of condensing scattered light that has been deflected by the observation object and passed through a diaphragm arranged at a position conjugate with the point light source to form a real image on the detection surface, and the point light source of 2 A scanning step in which the illumination step and the imaging step are performed while dimensionally scanning to generate four-dimensional data composed of two-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface, and the four-dimensional data are obtained from the two-dimensional coordinates of the light source surface. And a differential phase image acquisition step of acquiring a two-dimensional differential phase image of the observation object by calculating the barycentric integral in.
［装置構成］
　図１０は本実施形態の透過型顕微鏡装置１の構成例を示すブロック図である。前述した顕微鏡観察方法は、例えば、図１０に示すように、絞りを備えた光学系２と、結像した２次元像を検出する撮像部３と、光源走査などを制御する制御部４と、４次元画像データの蓄積、保管、計算処理を行うデータ処理部５を備える透過型顕微鏡装置１により実施することができる。光学系２には、光源と、例えば図１～図２に示す光学系を実現する複数のレンズを用いた結像系が設けられている。 [Device configuration]
FIG. 10 is a block diagram showing a configuration example of the transmission microscope apparatus 1 of this embodiment. In the above-described microscope observation method, for example, as shown in FIG. 10, an optical system 2 having a diaphragm, an imaging unit 3 for detecting a formed two-dimensional image, a control unit 4 for controlling light source scanning, and the like, This can be performed by the transmission microscope apparatus 1 including the data processing unit 5 that stores, stores, and calculates the four-dimensional image data. The optical system 2 is provided with a light source and an imaging system using a plurality of lenses that realize the optical system shown in FIGS. 1 and 2, for example.
　図１Ａに示すWaddellらが提案した収束ビーム照射４次元法と、図１Ｂに示す本発明の平行光（ビーム）照射４次元法とでは、絞りと観察対象物の配置が異なるが、本実施形態の透過型顕微鏡装置１の光学系２の基本構成は、Waddellらが提案した収束ビーム照射４次元法によるものと同じである。また、撮像部３には、２次元検出器を用いることができる。 The arrangement of the aperture and the observation object is different between the convergent beam irradiation four-dimensional method proposed by Waddell et al. Shown in FIG. 1A and the parallel light (beam) irradiation four-dimensional method of the present invention shown in FIG. 1B. The basic configuration of the optical system 2 of the transmission microscope apparatus 1 is the same as that of the convergent beam irradiation four-dimensional method proposed by Waddell et al. In addition, a two-dimensional detector can be used for the imaging unit 3.
　本実施形態の４次元顕微鏡は、STEM機能を持つ電子視顕微鏡において、光源走査という点では同じだが、物面上の観察対象物を収束ビームで走査する本来の方法を、平行光（ビーム）照射の照射方向を走査する方法に改変することで実現される。 The four-dimensional microscope of the present embodiment is the same as the electron microscope having the STEM function in terms of light source scanning, but the original method of scanning the observation object on the object surface with the convergent beam is a parallel light (beam) irradiation. It is realized by changing the method of scanning the irradiation direction of.
　以上詳述したように、本実施形態の顕微鏡観察方法は、平行光照明４次元法であり、実像を第一義的観察対象としているため、観察対象物の強度像及び微分位相像を、操作性良く取得することができる。また、本実施形態の透過型顕微鏡装置１は、STEM装置のSTEM機能を活用し、TEMとして使用することでも、TEMにSTEM機能を付加することでも実現できる。 As described in detail above, the microscope observation method of the present embodiment is a parallel light illumination four-dimensional method, and since the real image is the primary observation target, the intensity image and the differential phase image of the observation target are manipulated. It can be acquired well. Further, the transmission microscope apparatus 1 of the present embodiment can be realized by utilizing the STEM function of the STEM apparatus and using it as a TEM or by adding the STEM function to the TEM.
（データ処理方法）
＜ステップ１：４次元データ化＞
　以下、図１１を用いて４次元法におけるデータ処理の流れを説明する。観察対象物上の点を実座標ｒｄに対応させ、点光源の位置を周波数座標ｓｓに対応させる形でTEM実験を行ったとき観測される2次元実像データから４次元データ｜ＵＴＥＭ（ｓｓ，ｒｄ）｜２を構成する。 (Data processing method)
<Step 1: 4D data conversion>
The flow of data processing in the four-dimensional method will be described below with reference to FIG. A point on the observed object to correspond to the actual coordinates r d, 4-dimensional data the position of the point light source from the two-dimensional real image data to be observed when performing TEM experiments in a manner to correspond to the frequency coordinates s s | U TEM ( s s, r d) | constituting the two.
＜ステップ２：積分操作＞
　ステップ１で得られた４次元データを用い、個別ｒｄに対して2次元ｓｓ座標上の面積積分を行い、強度像を得る。並行して、ステップ１で得られた４次元データを用い、個別ｒｄに対して2次元ｓｓ座標上の重心積分を行い、即ちｓｓ座標に対し１次モーメントを計算し、それを強度像で除し、微分位相像を得る。 <Step 2: Integration operation>
Using 4-dimensional data obtained in step 1, performs area integration on the two-dimensional s s coordinates for individual r d, obtain intensity image. In parallel, using a four-dimensional data obtained in step 1, perform the centroid integration on the two-dimensional s s coordinates for individual r d, i.e. s s coordinates a moment calculated on the strength it Divide by the image to obtain the differential phase image.
（第２の実施形態）
　次に、本発明の第２の実施形態に係る４次元顕微鏡観察方法について説明する。通常のSTEM法でも良く知られているように、観察物上の収束ビーム走査を光源の走査で行う代わりに、光源を例えば光軸上に固定し、光源と共役位置にある観察物自体を移動走査しても同じ結果が得られる。TEMに対して同様のことを行うには光源と共役な位置にある絞りを移動走査すればよい。これを本発明である4D-TEMに適用すれば前述した第１の実施形態において光源走査ではなく、光源を固定し、代わって光源面の共役面上にある絞りを走査する方法を採用すると第２の実施形態となる。 (Second embodiment)
Next, a four-dimensional microscope observation method according to the second embodiment of the present invention will be described. As is well known in the ordinary STEM method, instead of scanning the focused beam on the observation object by scanning the light source, the light source is fixed on the optical axis, for example, and the observation object itself in a conjugated position with the light source is moved. The same result is obtained by scanning. To do the same for the TEM, move scan the aperture at the position conjugate with the light source. If this is applied to the 4D-TEM of the present invention, the method of fixing the light source instead of scanning the light source in the first embodiment and scanning the aperture on the conjugate plane of the light source surface instead of scanning the light source is adopted. 2 embodiment.
　走査法を光源走査から絞り走査に変える以外第１の実施形態との間に全く差がないので顕微鏡装置もデータ処理法も第１の実施形態と同じである。 Except that the scanning method is changed from light source scanning to aperture scanning, there is no difference from the first embodiment, so the microscope apparatus and data processing method are the same as in the first embodiment.
（第３の実施形態）
　この実施形態では、絞りは通常の円形ではなく、横長に直線状に延びたスリットである。そして、点光源またはスリットの走査をスリット長手方向に直交した１次元で行い、３次元データを生成する走査工程において、図５および図６に示すように、点光源を、観察試料が示す最大偏向をカバーするようなスリットの開口端を越える位置から両側対照的に開口全域を走査する。これを開口全走査3D-TEM法と呼ぶ。この次元縮減で観測効率が向上する。 (Third Embodiment)
In this embodiment, the diaphragm is not a normal circular shape, but a slit extending in a straight line in a horizontally long shape. Then, the scanning of the point light source or the slit is performed in one dimension orthogonal to the slit longitudinal direction, and in the scanning step of generating three-dimensional data, as shown in FIGS. The entire area of the opening is scanned symmetrically on both sides from a position beyond the opening end of the slit covering the slit. This is called the full aperture scanning 3D-TEM method. This reduction in dimension improves observation efficiency.
走査法を２次元走査から１次元走査に変える以外第１の実施形態との間に全く差がないので顕微鏡装置もデータ処理法も第１の実施形態と同じである。第２実施形態以降の説明では、第１実施形態と同一の構成についての説明を省略する。 The microscope apparatus and the data processing method are the same as those in the first embodiment because there is no difference from the first embodiment except that the scanning method is changed from two-dimensional scanning to one-dimensional scanning. In the description of the second and subsequent embodiments, the description of the same configuration as that of the first embodiment will be omitted.
（第４の実施形態）
　第１の実施形態から第２の実施形態への移行で説明し、かつ図５～８からも直感的に分かるように、光源点を走査するか絞りを走査するかは相対的なので第３の実施形態において１次光源点の１次元走査を絞り位置のｙ方向の１次元移動走査に置き換えても全く同じ３次元データの取得ができる。 (Fourth Embodiment)
As described in the transition from the first embodiment to the second embodiment, and as can be seen intuitively from FIGS. 5 to 8, it is relative whether to scan the light source point or the aperture, so that the third embodiment is used. In the embodiment, even if the one-dimensional scanning of the primary light source point is replaced with the one-dimensional moving scanning of the diaphragm position in the y direction, the same three-dimensional data can be acquired.
走査法を光源走査から絞り走査に変え同時に２次元走査から１次元走査に変える以外第１の実施形態との間に全く差がないので顕微鏡装置もデータ処理法も第１の実施形態と同じである。 Since there is no difference from the first embodiment except that the scanning method is changed from light source scanning to aperture scanning and from two-dimensional scanning to one-dimensional scanning at the same time, the microscope apparatus and the data processing method are the same as those in the first embodiment. is there.
（第５の実施形態）
　この実施形態では、スリットは横長に直線状に延びている。そして、点光源の走査を１次元で行い、３次元データを生成する走査工程において、点光源の走査領域を、スリットの開口部の中央部を除く、走査方向の両端部のうちの少なくともいずれか一方に限定している。これを開口端走査3D-TEM法と呼ぶ。 (Fifth Embodiment)
In this embodiment, the slit extends horizontally in a straight line. Then, in the scanning step of scanning the point light source in one dimension and generating three-dimensional data, the scanning region of the point light source is at least one of both ends in the scanning direction excluding the central portion of the opening of the slit. Limited to one side. This is called the aperture edge scanning 3D-TEM method.
　走査領域は、前記スリットと直交する光軸方向から見た平面視において、スリットにおける一対の開口端縁のうちの少なくともいずれか一方に対して、走査方向の両側にわたって画定されるとともに、走査領域の総和は、想定される最大微分位相量の半分となっている。 The scanning region is defined over both sides in the scanning direction with respect to at least one of the pair of opening edges in the slit in a plan view seen from the optical axis direction orthogonal to the slit, and the scanning region The total sum is half of the maximum expected differential phase amount.
　図７および図７８に示す例では、走査領域は、一対の開口端縁のうちの少なくともいずれか一方に対して、走査方向の両側にわたって均等に画定されている。
　前記走査領域は、両方の開口端縁に対して、前記走査方向の両側にわたって均等に画定されている。 In the example shown in FIGS. 7 and 78, the scanning region is evenly defined across both sides in the scanning direction with respect to at least one of the pair of aperture edges.
The scanning area is evenly defined across both sides of the scanning direction with respect to both open edge edges.
　図９は、図７および図８に示す両端両側開口端走査法６１（図９Ａ）のヴァリエーション例である。走査領域は、図９Ｂに示す片端両側開口端走査法６２ように、一方の開口端縁に対して、端縁の両側にわたって均等に画定されてもよい。
　また、図９Ｃに示す両端片側開口端走査Ｉ法６３、図９Ｄに示す両端片側開口端走II法６４のように、走査領域は、上下一対の開口端縁の両方に対して、それぞれ端縁の片側ずつに均等に画定されてもよい。 FIG. 9 is an example of a variation of both-ends both-sides opening-end scanning method 61 (FIG. 9A) shown in FIGS. 7 and 8. The scanning region may be evenly defined across both sides of one open edge with respect to one open edge, as in the single-ended bilateral open-end scanning method 62 shown in FIG. 9B.
Further, as in the double-ended single-sided open end scanning I method 63 shown in FIG. 9C and the double-ended single-sided open end running II method 64 shown in FIG. May be evenly defined on each side.
　ここで、本実施形態に測定方法における感度の定量比較について説明する。
　スリット絞りを用いる1次元光源走査を基礎とした4次元透過型電子顕微鏡で観測されるのは3次元データ、|ＵＴＥＭ(s, r) |2（sは1次元 rは2次元）である。このデータにおいて、空間座標rを固定したとき、s に関する1次元データにつき重心位置を計算することで微分位相∇θ(r)が求まる。この微分位相は、物理的には、電子・原子衝突に由来する対象物局所rでの電子線の屈折度合い、すなわち偏向角度に対応する。 Here, the quantitative comparison of sensitivity in the measuring method according to the present embodiment will be described.
Three-dimensional data, |U TEM (s, r) | 2 (s is one-dimensional r is two-dimensional) is observed by a four-dimensional transmission electron microscope based on one-dimensional light source scanning using a slit diaphragm. .. In this data, when the spatial coordinate r is fixed, the differential phase ∇θ (r) can be obtained by calculating the position of the center of gravity for the one-dimensional data related to s. This differential phase physically corresponds to the degree of refraction of the electron beam at the object local region r due to electron-atom collision, that is, the deflection angle.
　微分位相∇θ(r)を実験的に充分な精度で求めるには、電子線観測特有の雑音（ショットノイズ）に打ち勝たなくてはならない。すなわち電子線ショットノイズ由来の重心位置の揺らぎ（標準偏差）より∇θ(r)が大きくなければならない。この信号対雑音の問題を重心位置計算の特殊性に依拠して考え、信号である電子線偏向角を充分な空間分解能を持って観測するのに必要な電子線照射量につき理論計算を行う。 In order to obtain the differential phase ∇θ(r) experimentally with sufficient accuracy, it is necessary to overcome the noise (shot noise) peculiar to electron beam observation. That is, ∇θ(r) must be larger than the fluctuation (standard deviation) of the center of gravity position due to electron beam shot noise. This problem of signal-to-noise is considered based on the peculiarity of the calculation of the position of the center of gravity, and the theoretical calculation is carried out for the electron beam dose necessary for observing the electron beam deflection angle, which is a signal, with sufficient spatial resolution.
　4つの量、観察対象物の電子線偏向角度、電子線照射量、空間分解能、微分位相分解能（偏向角度の観察精度で像コントラストの観測精度に対応）の関係は、一般に開口全走査（光源全域走査）4D-TEMと開口端走査（光源部分域走査）4D-TEMでは異なり、電子線照射量に上限のある蛋白質やDNAのような生体分子の電顕観察において、部分的電子線走査を行う開口端走査4D-TEMが有利となる。以下、次元縮減のために横長スリットを用いた3D-TEM法に対し感度向上を詳説する。 The relationship among the four quantities, the electron beam deflection angle of the observation object, the electron beam irradiation amount, the spatial resolution, and the differential phase resolution (corresponding to the observation accuracy of the image contrast by the observation accuracy of the deflection angle) is generally the full aperture scan (the entire light source area). Scanning) 4D-TEM and open edge scanning (light source partial area scanning) 4D-TEM is different, and partial electron beam scanning is performed in electron microscopic observation of biological molecules such as proteins and DNA that have an upper limit on electron beam irradiation. Open edge scanning 4D-TEM is advantageous. In the following, the sensitivity improvement for the 3D-TEM method using a horizontally long slit for dimension reduction will be described in detail.
　図５および図６に開口全走査3D-TEM実験における電子線偏向がない場合（A）とある場合（B）の模式図を示す。この図は、光源面に光源と共役な位置にあるスリット絞りを投影し、模式的に１次元走査を行う光源面sy軸上に光源走査点を表示している。図１Aの斜線部は、観察対象物局所rにおいて電子線偏向がない場合、すなわち∇θ(r) =0のときの、スリット絞りによる遮蔽なく電子線が検出器に届き像が観察される範囲である。 FIG. 5 and FIG. 6 are schematic diagrams showing a case without electron beam deflection (A) and a case with electron beam deflection (B) in the full aperture scanning 3D-TEM experiment. In this figure, a slit diaphragm located at a position conjugate with the light source is projected on the light source surface, and the light source scanning points are displayed on the light source surface s y axis for schematically performing one-dimensional scanning. The shaded area in Fig. 1A is the range in which the electron beam reaches the detector without being blocked by the slit diaphragm when there is no electron beam deflection in the observation object local area r, that is, when ∇θ(r) = 0. Is.
　図６の斜線部は、観察対象物局所rにおいて電子線偏向がある場合、すなわち∇θ (r) ＜0のとき、スリット絞りに遮蔽されずに電子線が検出器に届き像が観察される範囲である。図６のスリット上方では偏向によりスリットを迂回した電子線が検出器に届き観測され、下方では逆に、図５では観測された電子線がスリットにより遮蔽され観測されないことを表している。
　開口の範囲は[-L/2, L/2]（Lは整数）だが、走査範囲はl/4のマージンを取り[-L/2-l/4, L/2+l/4]（L, l共に整数）となり、走査幅はL+ l/2である。重心移動に対応する微分位相の大きさ|∇θ(r)|が走査マージンl/4より小さくなることが重要である。 The shaded area in FIG. 6 shows the image when the electron beam is deflected at the local r of the observation object, that is, when ∇θ (r) <0, the electron beam reaches the detector without being blocked by the slit diaphragm and the image is observed. It is a range. Above the slit in FIG. 6, an electron beam bypassing the slit due to deflection reaches the detector and is observed, and conversely, in FIG. 5, the observed electron beam is blocked by the slit and is not observed.
The aperture range is [-L/2, L/2] (L is an integer), but the scanning range has a margin of l/4 [-L/2-l/4, L/2+l/4] ( Both L and l are integers), and the scan width is L+l/2. It is important that the magnitude |∇θ(r)| of the differential phase corresponding to the movement of the center of gravity is smaller than the scanning margin l/4.
　図７および図８に開口端走査3D-TEMにおける電子線偏向がない場合（A）とある場合（B）の模式図を示す。図５および図６の開口全走査3D-TEMとの違いは、走査範囲が開口端近傍に限定されることで、そのため走査範囲は、[-L/2-l/4, -L/2+l/4] と[L/2-l/4, L/2+l/4]に二分しかつ狭まり（L,l共に整数）、全走査幅はlとなる。一般にlはLに比べ極めて小さいので、全照射電子線量に制限がある場合、電子線を狭い走査範囲lに集中できる開口端走査3D-TEMがショットノイズ由来の雑音を軽減でき、結果的に高感度化可能である。 FIGS. 7 and 8 show schematic diagrams in the case where there is no electron beam deflection in the aperture edge scanning 3D-TEM (A) and the case (B). The difference from the full aperture scanning 3D-TEM in FIGS. 5 and 6 is that the scanning range is limited to the vicinity of the aperture end. Therefore, the scanning range is [-L/2-l/4, -L/2+ l/4] and [L/2-l/4, L/2+l/4] are bisected and narrowed (both L and l are integers), and the total scan width is l. In general, l is extremely small compared to L, so when the total irradiation electron dose is limited, open-edge scanning 3D-TEM, which can concentrate the electron beam in a narrow scanning range l, can reduce the noise derived from shot noise, resulting in high. Sensitivity is possible.
　図５から図８の各斜線部領域に照射される全照射電子量をVとすると、これは４D STEMにおいて観察対象物局所のrに照射される全電子線量に対応し、その照射範囲δｄ　は分解能に相当する。電子線照射量Dは一般に単位面積当たりの電子線数で表されるので、照射範囲δｄに当たる　電子線量Vは下記（１７）式で与えられる。
V= Dδｄ 2　　　　　　　　　　　　　　　（１７） Letting V be the total irradiation electron amount irradiated to each hatched area in FIGS. 5 to 8, this corresponds to the total electron dose irradiated to r in the observation object local area in 4D STEM, and its irradiation range δ d Corresponds to the resolution. Since the electron beam dose D is generally represented by the number of electron beams per unit area, the electron dose V corresponding to the irradiation range δ d is given by the following equation (17).
V= Dδ d 2 (17)
　低温電子顕微鏡を用いた場合の蛋白質の照射限界量Dlimitは、10-30 電子/Å２[1]、DNAのそれは50電子/Å２[2]から500電子/Å２[3]といわれている。したがってδｄÅの分解能を得ようと思えば、例えば蛋白質ではVlimit＝20δｄ 2 　電子が、DNAではVlimit＝200δｄ 2 　電子が限界照射電子量となる。逆にVlimitが与えられれば、δｄÅの分解能に制限される。 It is said that the D limit of protein irradiation when using a cryo-electron microscope is 10-30 electrons / Å 2 [1], and that of DNA is 50 electrons / Å 2 [2] to 500 electrons / Å 2 [3]. ing. Therefore if you going to get the resolution of [delta] d Å, for example, V limit = 20δ d 2 electrons in protein, V limit = 200δ d 2 electrons in DNA is critical irradiation amount of electrons. Conversely, if V limit is given, the resolution is limited to δ d Å.
　モノによる電子線の偏向は屈折現象なので、その大きさは電子線の真空との差屈折率Δｎ(r)に比例する。この差屈折率Δｎ(r)を用いて、位相は下記（１８）式で得られる。
　　θ (r)＝2πΔｎ(r)ｄ/λ　　　　　　　　　　　　　（１８）
（Δｎ(r)：局所の差屈折率、ｄ ：rにおけるモノの厚さ、λ：電子線波長） Since the deflection of the electron beam by the object is a refraction phenomenon, its magnitude is proportional to the differential refractive index Δn(r) of the electron beam with respect to the vacuum. Using this differential refractive index Δn (r), the phase can be obtained by the following equation (18).
θ(r)=2πΔn(r)d/λ (18)
(Δn(r): local differential refractive index, d: thickness of the object at r, λ: electron beam wavelength)
　原子オーダーの厚さの物質を考えるとs座標の重心である微分位相は下記（１９）式で与えられる。
∇θ(r)-＝（2π/λ）・（∇Δｎ (r)ｄ）≒（2π/λ）Δｎ(r)　（１９） Considering a substance with an atomic thickness, the differential phase, which is the center of gravity of the s coordinate, is given by the following equation (19).
∇θ (r)-= (2π / λ) ・ (∇Δn (r) d) ≒ (2π / λ) Δn (r) (19)
　一方∇θ (r) は、 3次元データ、|ＵＴＥＭ(s, r) |2のsと同じ次元を持ちかつsのsy軸は下記（２０）式から（２１）式で定義される。
　　sy＝（2π/λ）・（a/ｆ）　　　　　　　　　　　　　（２０）
（a　：スリット絞りsy軸の実寸、ｆ　：レンズの焦点距離）
　ここで、a/ｆ　は絞り座標syの検出器から見た方角なので、（１９）式中のΔｎ(r)がsy軸の電子線偏向角に相当することがわかる。またスリット絞りの端の実寸をaoとすれば、（２０）式を用いてスリット端座標syoは以下となり、ao /ｆ　はいわゆる対物レンズの開口数となる。
　　　syo＝（2π/λ）・（ao /ｆ）　　　　　　　（２１）
開口数ao /ｆ　はまた顕微鏡の物理的分解能δpと以下の関係で結ばれる。
　　δp＝λ/（ao /ｆ）　　　　　　　　　　　　 (２２) On the other hand, ∇θ (r) has the same dimension as s of 3D data, |U TEM (s, r) | 2 , and the s y axis of s is defined by the following equations (20) to (21). ..
s y =(2π/λ)・(a/f) (20)
(A: slit stop s y- axis actual size, f: lens focal length)
Since a / f is a direction seen from the detector aperture coordinate s y, it can be seen that (19) [Delta] n in formula (r) corresponds to the electron beam deflection angle of s y-axis. If the actual size of the end of the slit diaphragm is a o , the slit end coordinates s yo are as follows using Eq. (20), and a o / f is the so-called numerical aperture of the objective lens.
s yo = (2π/λ)・(a o /f) (21)
The numerical aperture a o / f is also connected to the physical resolution δ p of the microscope in the following relationship.
δ p =λ/(a o /f) (22)
　以上の数式を基に、2つの3D-TEM法におけるショットノイズ由来の重心位置の揺らぎ（標準偏差）を算出する、前述したように、この量の大きさが手法の感度を決める。標準偏差が大きいほど重心位置決め精度が落ちる。精度を高めるため、標準偏差を小さくするには、下記にも示すように多量の電子線照射が必要で生体分子には適用できない。 Based on the above formula, the fluctuation (standard deviation) of the position of the center of gravity derived from shot noise in the two 3D-TEM methods is calculated. As mentioned above, the magnitude of this amount determines the sensitivity of the method. The larger the standard deviation, the lower the center of gravity positioning accuracy. In order to improve the accuracy, in order to reduce the standard deviation, a large amount of electron beam irradiation is required as shown below and it cannot be applied to biomolecules.
　まず前提として図５から図８で示す斜線部分の電子線入力は一様であり、各点（各ピクセル）に来る電子数はある平均値（これはピクセルの位置に寄らず同一とする。これが一様入力の意味）の周りにポアッソン分布するとする（ショットノイズ仮定）。 First, as a premise, the electron beam input in the shaded areas shown in FIGS. 5 to 8 is uniform, and the number of electrons coming to each point (each pixel) is a certain average value (this is the same regardless of the pixel position. (Meaning uniform input) around the Poisson distribution (shot noise assumption).
　このとき斜線部分の重心は本来長方形の中心だが、ショットノイズのためその中心位置から揺らぐ。この揺らぎ、正確には標準偏差は、斜線部に来る電子線総数が大きいほど小さくなる。具体的に標準偏差は、図５および図６の開口全走査3D-TEMのケースでは（２３）式、図７および図８の開口端走査3D-TEMのケースでは（２４）式で表される。
　σｗ＝L /（12Vｗ) 1/2 　　　　　　　　　（２３）
(Vｗは[-L/2-l/4, L/2+l/4]範囲で走査照射した電子線総数)
σe＝l / (24Ve) 1/2　 　　　　　　　　　　（２４）
(Veは[-L/2-l/4, -L/2+l/4] と[L/2-l/4, L/2+l/4]での電子線総数) At this time, the center of gravity of the shaded area is originally the center of the rectangle, but it fluctuates from the center position due to shot noise. This fluctuation, to be exact, the standard deviation becomes smaller as the total number of electron beams coming to the shaded area increases. Specifically, the standard deviation is expressed by equation (23) in the case of full aperture scanning 3D-TEM in FIGS. 5 and 6, and by equation (24) in the case of aperture edge scanning 3D-TEM in FIGS. 7 and 8. ..
σ w =L/(12V w ) 1/2 (23)
( Vw is the total number of electron beams scanned and irradiated in the [-L/2-l/4, L/2+l/4] range)
σ e =l / (24V e ) 1/2 (24)
(V e is the total number of electron beams at [-L / 2-l / 4, -L / 2 + l / 4] and [L / 2-l / 4, L / 2 + l / 4])
　図５から図８の比較からわかるように Ｌ≫lなので、同じ電子線総数（Vｗ＝Ve）ならσｗ≫σe　となり、逆にσｗ＝σe　と感度をそろえるなら、開口端走査3D-TEMでの電子線総数Veは開口全走査3D-TEM Vｗに較べ微小ですむ。これは生体分子のように限界電子線数が小さい観察物に対し有利となり、限界電子線数内での高分解能化を可能とする。 As can be seen from the comparison of FIGS. 5 to 8, since L >> l, if the total number of electron beams (V w = V e ) is the same, then σ w >> σ e , and conversely, if the sensitivity is the same as σ w = σ e , the opening end The total number of electron beams V e in the scanning 3D-TEM is smaller than that in the full aperture scanning 3D-TEM V w . This is advantageous for an observation object such as a biomolecule having a small limiting electron beam number, and enables high resolution within the limiting electron beam number.
　ここでL,　l　の大きさを見積もる。重心観測の要請から、光源の走査範囲において観察される重心移動対応の偏向角Δｎ(r)をどの細かさで観測するのかがまず問題となる。そこで偏向角の平均値のN等分の精度で求めるとしよう。次に観測範囲での最大の偏向角を平均値のＭ倍としよう。結局広がりのある∇θ(r) の観測点はMN点必要になる。これが図５から図８における重心変動域のl/4に対応するので、lの大きさは（２５）式で与えられる。
l＝４MN　　　　　　　　　　　　　　（２５） Here, the size of L, l is estimated. Due to the request for the observation of the center of gravity, the first problem is how fine the deflection angle Δn(r) corresponding to the movement of the center of gravity observed in the scanning range of the light source is to be observed. Therefore, let's assume that the average deflection angle is calculated with N equal accuracy. Next, let the maximum deflection angle in the observation range be M times the average value. Eventually, MN points are required for the observation points of ∇θ(r) with a spread. Since this corresponds to l/4 of the center-of-gravity variation region in FIGS. 5 to 8, the size of l is given by equation (25).
l=4MN (25)
　他方、Lを求めるには、光源の走査範囲と偏向角平均値の比が必要になる。角度で表すと、走査範囲は（２１）式に示す2ao /ｆ となり、偏向角平均値はΔｎ(r)の平均値となる。また（２２）式に従えばao /ｆ は開口数として物理分解能に関係する。 On the other hand, in order to obtain L, the ratio between the scanning range of the light source and the average deflection angle value is required. Expressed in terms of angle, the scanning range is 2a o / f shown in Eq. (21), and the average deflection angle is the average value of Δn (r). Further, according to the equation (22), a o /f is related to the physical resolution as a numerical aperture.
　例えば観測波長λの1/100の分解能がほしければ(100 kV電顕の場合、波長は0.037Åなので1/100の分解能は3.7Åとなり、原子分解能に近づく）ao /ｆ＝1/100となるが、生体分子を構成する炭素、酸素、窒素のような軽元素の差屈折率Δｎ(r) は１０－４程度なので、比は100倍。Δｎ(r)をNの精度で求めるので、Ｌは以下となる。
　L=200N　　　　　　　　　　　　　　　　　　（２６） For example, if you want a resolution of 1/100 of the observed wavelength λ (in the case of a 100 kV electron microscope, the wavelength is 0.037Å, the resolution of 1/100 is 3.7Å, which is close to the atomic resolution) a o /f=1/100 However, the differential refractive index Δn (r) of light elements such as carbon, oxygen, and nitrogen that make up the biomolecule is about 10 -4 , so the ratio is 100 times. Since Δn(r) is obtained with an accuracy of N, L is as follows.
L=200N (26)
　観測で、標準偏差が1ピクセルまで許容されると考えると、（２３）、（２４）においてσｗ＝σe＝1と置き、分解能と電子線総数を結ぶ(１)式およびＬ, lを与える（２５）、（２６）式と結合すると、それぞれの手法で決まる分解能 δw,　、δeが下記（２７）式、（２８）式のように求まる。
　開口全走査3D-TEM：δw＝L/(12Dlimit) 1/2 ＝200N /(12Dlimit) 1/2  (２７)
　開口端走査3D-TEM：δe＝l/(24Dlimit) 1/2  ＝４MN/(24Dlimit) 1/2  (２８) Considering that the standard deviation is allowed up to 1 pixel in the observation, set σ w =σ e =1 in (23) and (24), and formula (1) connecting the resolution and the total number of electron beams and L, l When combined with the given equations (25) and (26), the resolutions δ w , δ e determined by each method can be obtained as the following equations (27) and (28).
Aperture full scan 3D-TEM: δ w =L/(12D limit ) 1/2 =200N /(12D limit ) 1/2 (27)
Aperture edge scan 3D-TEM: δ e =l/(24D limit ) 1/2 =4MN/(24D limit ) 1/2 (28)
　ここでM ＝N＝5とし、蛋白質のDlimit＝20電子/Å、DNAのDlimit＝200電子/Åとしたときの到達分解能を求めると以下になる。
　開口全走査3D-TEM：　蛋白質分解能＝64.5Å　ＤＮＡ分解能＝20.4Å　　（２９）
　開口端走査3D-TEM：　蛋白質分解能＝4.6Å　ＤＮＡ分解能＝1.4Å　　　（３０） Here, the ultimate resolution when M = N = 5, protein D limit = 20 electrons / Å, and DNA D limit = 200 electrons / Å is calculated as follows.
Full aperture scanning 3D-TEM: Protein resolution = 64.5Å DNA resolution = 20.4Å (29)
Open end scanning 3D-TEM: Protein resolution = 4.6Å DNA resolution = 1.4Å (30)
　上記の結果は、３次元版の4D-TEMでの感度向上の例であるが、結果は４次元版でも同じと考えて良いので、開口端走査4D-TEMの到達分解能が通常の開口全走査4D-TEMに比し10倍以上良く、原子分解能を実現できることがわかる。無論開口全走査4D-TEMですら従来の透過型電子顕微鏡での到達分解能に比べ遜色はなく、高感度のゼルニケ位相差電子顕微鏡の結果と同等の到達分解能である。 The above result is an example of sensitivity improvement in the 3D version of 4D-TEM, but since the result can be considered to be the same in the 4D version, the ultimate resolution of the aperture end scanning 4D-TEM is normal aperture full scanning. It can be seen that atomic resolution can be achieved, which is 10 times better than 4D-TEM. Of course, even the aperture full-scan 4D-TEM is comparable to the resolution achieved by the conventional transmission electron microscope, and the resolution is equivalent to the result of the highly sensitive Zernike phase contrast electron microscope.
（第６の実施形態）
　第１の実施形態１から第２の実施形態への移行で説明し、かつ図５～８からも直感的に分かるように、光源点を走査するか絞りを走査するかは相対的なので第６５の実施形態において光源点の1次元走査を絞り位置のｙ方向の１次元移動走査に置き換えても全く同じ３次元データの取得ができる。これが第６の実施形態であり、そのほか説明は第５の実施形態を援用する。 (Sixth Embodiment)
As described in the transition from the first embodiment to the second embodiment, and as can be seen intuitively from FIGS. 5 to 8, it is relative whether to scan the light source point or the aperture, and therefore the sixty-fifth. In the embodiment, even if the one-dimensional scanning of the light source points is replaced with the one-dimensional moving scanning of the aperture position in the y direction, the same three-dimensional data can be acquired. This is the sixth embodiment, and other explanations apply to the fifth embodiment.
（第７の実施形態）
　図３Ａで説明したように、4D-STEMにおいても従来法を凌駕する感度を絞り中央に遮蔽板を置くことで実現できる。この形態の絞りは開口部が環状（ドーナツ状）となるのですでに環状絞りとして通常STEMにおいてすでに活用されている（非特許文献１６）。しかし環状絞りを4D-STEMに応用した例は報告されていないのでこれを第７の実施形態とする。 (Seventh embodiment)
As described with reference to FIG. 3A, even in 4D-STEM, a sensitivity that surpasses that of the conventional method can be realized by placing a shielding plate in the center of the diaphragm. Since the opening of this type of diaphragm is annular (doughnut-shaped), it has already been used as an annular diaphragm in ordinary STEM (Non-Patent Document 16). However, since an example in which the annular diaphragm is applied to 4D-STEM has not been reported, this will be referred to as the seventh embodiment.
　１　顕微鏡装置
　２、１０、１００　光学系
　３　撮像部
　４　制御部
　５　データ処理部
　１１～１３、１０１～１０３　レンズ
　２１、１１１　光源面
　２２、１１３　物面
　２３、１１２　絞り面
　２４、１１４　検出面
２６　偏向コイル
３１　重心移動のない検出面/光源面明視野円盤
３２　重心移動のある検出面/光源面明視野円盤
４１　4D-TEMにおける光源の部分走査領域
４２　4D-STEMに用いられる環状絞り
４３　4D-STEMに用いられる電子線遮蔽板
５１　絞り開口
５２　横長スリット（絞り）
６０　１次元走査方向
６１　両端両側開口端走査法
６２　片端両側開口端走査法
６３　両端片側開口端走査I法
６４　両端片側開口端走査II法 1 Microscope device 2, 10, 100 Optical system 3 Imaging unit 4 Control unit 5 Data processing unit 11 to 13, 101 to 103 Lens 21, 111 Light source surface 22, 113 Object surface 23, 112 Aperture surface 24, 114 Detection surface 26 Deflection Coil 31 Detection surface without movement of center of gravity / light source surface bright field disk 32 Detection surface with movement of center of gravity / light source surface bright field disk 41 4 Partial scanning area of light source in D-TEM 42 4 Circular diaphragm used for D-STEM 43 4D-STEM Electron beam shielding plate 51 used for a diaphragm aperture 52 Horizontal slit (aperture)
60 one-dimensional scanning direction 61 both ends both sides opening end scanning method 62 one end both sides opening end scanning method 63 both ends one side opening end scanning I method 64 both ends one side opening end scanning II method

Claims (13)

  1. 　点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明工程と、
    　前記観察対象物で偏向され、前記点光源と共役な位置に配置された絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、
    　前記点光源を２次元走査しながら前記照明工程と前記結像工程を行い、光源面２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、
    　前記４次元データにおいて、前記光源面２次元座標における光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する顕微鏡観察方法。     An illumination step of irradiating the light emitted from the point light source to the observation object in parallel from various angles.
    An imaging step in which scattered light that is deflected by the observation object and passes through a diaphragm arranged at a position conjugate with the point light source is collected to form a real image on the detection surface.
    A scanning step of performing the illumination step and the imaging step while scanning the point light source in two dimensions to generate four-dimensional data composed of two-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface.
    Differential phase image acquisition step of acquiring a two-dimensional differential phase image of the observation object by calculating the barycentric integral of the light source surface bright field disk in the two-dimensional coordinates of the light source surface in the four-dimensional data. Method.
  2. 　点光源から射出された光を観察対象物に平行照射する照明工程と、
    　前記観察対象物で偏向され、前記点光源と共役な位置に配置された絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、
    　前記絞りを２次元走査しながら前記照明工程と前記結像工程を行い、走査２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、
    　前記４次元データにおいて、前記光源面２次元座標における光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する顕微鏡観察方法。     An illumination step of irradiating the light emitted from the point light source in parallel with the observation object,
    An imaging step in which scattered light that is deflected by the observation object and passes through a diaphragm arranged at a position conjugate with the point light source is collected to form a real image on the detection surface.
    A scanning step of performing the illumination step and the imaging step while scanning the aperture two-dimensionally to generate four-dimensional data composed of two-dimensional scanning coordinates and two-dimensional detection surface coordinates.
    Microscopic observation including a differential phase image acquisition step of calculating the integral of the center of gravity of the light source surface bright field disk at the light source surface two-dimensional coordinates in the four-dimensional data and acquiring the two-dimensional differential phase image of the observation object. Method.
  3. 　点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明工程と、
    　前記観察対象物で偏向され、前記点光源と共役な位置に配置された横長スリットである絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、
    　前記点光源の走査を横長スリットの長手方向に直交した１次元で行い、光源面1次元座標と検出面2次元座標からなる３次元データを生成する走査工程と、
    　前記３次元データにおいて、前記光源面１次元座標における１次元光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する顕微鏡観察方法。     An illumination step of irradiating the light emitted from the point light source to the observation object in parallel from various angles.
    An imaging step of collecting scattered light that has passed through a diaphragm, which is a horizontally long slit arranged at a position conjugate with the point light source and deflected by the observation object, to form a real image on the detection surface.
    A scanning step of scanning the point light source in one dimension orthogonal to the longitudinal direction of the lateral slit, and generating three-dimensional data composed of one-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface;
    The three-dimensional data includes a differential phase image acquisition step of calculating the integration of the center of gravity of the one-dimensional light source surface bright-field disk at the one-dimensional coordinates of the light source surface to acquire a two-dimensional differential phase image of the observation object. Microscopic observation method.
  4. 　点光源から射出された光を観察対象物に平行照射する照明工程と、
    　前記観察対象物で偏向され、前記点光源と共役な位置に配置された横長スリットである絞りを通過した散乱光を集光して検出面に実像を結像させる結像工程と、
    　前記絞りの走査を横長スリットの長手方向に直交した１次元で行い、光源面1次元座標と検出面2次元座標からなる３次元データを生成する走査工程と、
    　前記３次元データにおいて、前記光源面１次元座標における１次元光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する顕微鏡観察方法。     An illumination step of irradiating the light emitted from the point light source in parallel with the observation object,
    An image forming step of forming a real image on a detection surface by collecting scattered light that has been deflected by the observation object and has passed through a diaphragm that is a horizontally elongated slit arranged at a position conjugate with the point light source,
    A scanning step of scanning the diaphragm in one dimension orthogonal to the longitudinal direction of the lateral slit, and generating three-dimensional data consisting of one-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface;
    A differential phase image obtaining step of obtaining a two-dimensional differential phase image of the observation object by calculating the barycentric integral of the one-dimensional light source surface bright field disc in the one-dimensional coordinates of the light source surface in the three-dimensional data. Microscopic observation method.
  5. 　前記走査領域は、前記スリットと直交する光軸方向から見た平面視において、前記スリットにおける一対の開口端縁の両方に対して、前記スリット端縁の両側にわたって画定されるとともに、前記走査領域の総和は、想定される最大微分位相量の４倍近傍となることを特徴とする請求項３又は４に記載の顕微鏡観察方法。 The scanning region, in a plan view seen from the optical axis direction orthogonal to the slit, is defined over both sides of the slit edge with respect to both of the pair of opening edges in the slit, and The microscope observation method according to claim 3 or 4, characterized in that the sum is close to four times the assumed maximum differential phase amount.
  6. 　前記走査領域は、一対の前記開口端縁のうちのいずれか一方に対して、前記スリット端縁の両側にわたって画定されるとともに、前記走査領域の総和は、想定される最大微分位相量の２倍近傍となることを特徴とする請求項３又は４に記載の顕微鏡観察方法。 The scanning area is defined over both sides of the slit edge with respect to any one of the pair of opening edges, and the total of the scanning areas is twice the assumed maximum differential phase amount. 5. The microscope observing method according to claim 3, wherein the method is in the vicinity.
  7. 　前記走査領域は、一対の前記開口端縁のうちの両方の開口端縁に対して、スリットの開口内側または開口外側の片側ずつに画定されるとともに、前記走査領域の総和は、想定される最大微分位相量の２倍近傍となることを特徴とする請求項３又は４に記載の顕微鏡観察方法。 The scanning area is defined on both sides of the opening edge of the slit with respect to both of the opening edges of the pair of opening edges, and the sum of the scanning areas is the maximum assumed. The microscope observation method according to claim 3 or 4, wherein the amount is close to twice the differential phase amount.
  8. 　点光源から射出された光を、環状絞りを透過させながら観察対象物に収束ビームとして照射する照明工程と、
    　前記観察対象物で偏向された収束ビーを前記環状絞りと共役な位置に配置された検出面に回折像として結像させる結像工程と、
    　前記収束ビームを２次元走査することで得られる光源面２次元座標と検出面２次元座標からなる４次元データを生成する走査工程と、
    　前記４次元データにおいて、検出面２次元座標の環状絞りに対応する部分域での２次元データの重心積分を計算して、前記観察対象物の２次元微分位相像を取得する微分位相像取得工程と、を有する顕微鏡観察方法。     A lighting process in which the light emitted from a point light source is irradiated to the observation object as a convergent beam while passing through an annular diaphragm.
    An image forming step of forming a convergent beam deflected by the observation object as a diffraction image on a detection surface arranged at a position conjugate with the annular diaphragm,
    A scanning step of generating four-dimensional data composed of two-dimensional coordinates of a light source surface and two-dimensional coordinates of a detection surface obtained by two-dimensionally scanning the convergent beam;
    In the four-dimensional data, a differential phase image acquisition step of calculating a two-dimensional differential phase image of the observation object by calculating the barycentric integral of the two-dimensional data in the partial area corresponding to the annular diaphragm of the two-dimensional coordinates of the detection surface. And a microscope observing method having:
  9. 　点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明光学系と、光量を調整するために前記点光源と共役な位置に絞りを配置する絞り部と、観察対象物を通過した光を集光して検出面に実像を結像させる結像光学系と、
    　前記点光源を２次元走査しながら前記平行照射と前記結像を行う制御部と、光源面２次元座標と検出面２次元座標からなる４次元データを生成するデータ生成部と、
    　前記４次元データにつき、前記光源面２次元座標における重心積分を計算して、前記観察対象物の２次元微分位相像を取得するデータ処理部と、を有する透過型顕微鏡装置。     An illumination optical system that irradiates the observation target with light emitted from a point light source in parallel from various angular directions, a diaphragm portion that arranges a diaphragm at a position conjugate with the point light source in order to adjust the amount of light, and an observation target. An imaging optical system that collects the light that has passed through and forms a real image on the detection surface.
    A control unit that performs parallel irradiation and the image formation while scanning the point light source in two dimensions, and a data generation unit that generates four-dimensional data composed of two-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface.
    A transmissive microscope device having a data processing unit that calculates the integral of the center of gravity in the two-dimensional coordinates of the light source surface for the four-dimensional data and acquires a two-dimensional differential phase image of the observation object.
  10. 点光源から射出された光を観察対象物に平行照射する照明光学系と、
    　光量を調整するために前記点光源と共役な位置に絞りを配置する絞り部と、観察対象物を通過した光を集光して検出面に実像を結像させる結像光学系と、
    　前記絞りを２次元走査しながら前記平行照射と前記結像を行う制御部と、走査２次元座標と検出面２次元座標からなる４次元データを生成するデータ生成部と、
    　前記４次元データにつき、前記走査２次元座標と点対称の光源面２次元座標における重心積分を計算して、前記観察対象物の２次元微分位相像を取得するデータ処理部と、を有する透過型顕微鏡装置。     An illumination optical system that irradiates the light emitted from the point light source to the observation object in parallel,
    A diaphragm portion that arranges a diaphragm at a position conjugate with the point light source in order to adjust the amount of light, and an imaging optical system that collects light that has passed through an observation object and forms a real image on the detection surface.
    A control unit that performs the parallel irradiation and the image formation while two-dimensionally scanning the diaphragm, and a data generation unit that generates four-dimensional data composed of scanning two-dimensional coordinates and detection surface two-dimensional coordinates,
    A transmission type having a data processing unit that calculates the integral of the center of gravity of the four-dimensional data at the two-dimensional coordinates of the light source surface that is point-symmetrical to the two-dimensional coordinates of the scan and acquires the two-dimensional differential phase image of the observation object. Microscope device.
  11. 　点光源から射出された光を観察対象物にいろいろな角度方向から平行照射する照明光学系と、
    　光量を調整するために前記点光源と共役な位置に横長スリットである絞りを配置する絞り部と、観察対象物を通過した光を集光して検出面に実像を結像させる結像光学系と、
    　前記点光源の走査を横長スリットの長手方向に直交した１次元で行い、光源面1次元座標と検出面2次元座標からなる３次元データを生成するデータ生成部と、
    　前記３次元データにおいて、前記光源面１次元座標における１次元光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得するデータ処理部と、を有する透過型顕微鏡装置。     An illumination optical system that irradiates the observation object in parallel with the light emitted from a point light source from various angular directions.
    A diaphragm portion that arranges a diaphragm that is a horizontally long slit at a position conjugate with the point light source in order to adjust the amount of light, and an imaging optical system that collects light that has passed through an observation object and forms a real image on the detection surface. When,
    A data generator that scans the point light source in one dimension orthogonal to the longitudinal direction of the lateral slit, and generates three-dimensional data composed of one-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface;
    In the three-dimensional data, a transmission type having a data processing unit that calculates the integral of the center of gravity of the one-dimensional light source surface bright-field disk at the one-dimensional coordinates of the light source surface and acquires a two-dimensional differential phase image of the observation object. Microscope device.
  12. 　点光源から射出された光を観察対象物に平行照射する照明光学系と、
    　光量を調整するために前記点光源と共役な位置に横長スリットである絞りを配置する絞り部と、観察対象物を通過した光を集光して検出面に実像を結像させる結像光学系と、
    　前記絞りの走査を横長スリットの長手方向に直交した１次元で行い、光源面1次元座標と検出面2次元座標からなる３次元データを生成するデータ生成部と、
    　前記３次元データにおいて、前記光源面１次元座標における１次元光源面明視野円盤の重心積分を計算して、前記観察対象物の２次元微分位相像を取得するデータ処理部と、を有する透過型顕微鏡装置。     An illumination optical system that irradiates the light emitted from the point light source to the observation object in parallel,
    A diaphragm portion that arranges a diaphragm that is a horizontally long slit at a position conjugate with the point light source in order to adjust the amount of light, and an imaging optical system that collects light that has passed through an observation object and forms a real image on the detection surface. When,
    A data generator that scans the diaphragm in one dimension orthogonal to the longitudinal direction of the lateral slit, and generates three-dimensional data composed of one-dimensional coordinates of the light source surface and two-dimensional coordinates of the detection surface;
    In the three-dimensional data, a transmission type having a data processing unit that calculates the integral of the center of gravity of the one-dimensional light source surface bright-field disk at the one-dimensional coordinates of the light source surface and acquires a two-dimensional differential phase image of the observation object. Microscope device.
  13. 　点光源から射出された光を、環状絞りを透過させながら収束ビームとして観察対象物に照射する照明工学系と、
    　前記観察対象物で偏向された収束ビーを前記環状絞りと共役な位置に配置された検出面に回折像として結像させる結像工学系と、
    　前記収束ビームを２次元走査することで得られる光源面２次元座標と検出面２次元座標からなる４次元データを生成するデータ生成部
    と、
    　前記４次元データにおいて、検出面２次元座標の環状絞りに対応する部分域での２次元データの重心積分を計算して、前記観察対象物の２次元微分位相像を取得するデータ処理部と、を有する走査透過型顕微鏡装置。     An illumination engineering system that irradiates the observation target with light emitted from a point light source as a convergent beam while passing through an annular diaphragm.
    An imaging engineering system that forms a convergent beam deflected by the observation object as a diffraction image on a detection surface arranged at a position conjugate with the annular diaphragm,
    A data generator that generates four-dimensional data composed of two-dimensional coordinates of a light source surface and two-dimensional coordinates of a detection surface obtained by two-dimensionally scanning the convergent beam;
    In the four-dimensional data, a data processing unit that calculates a two-dimensional differential phase image of the observation object by calculating the barycentric integral of the two-dimensional data in the partial area corresponding to the annular diaphragm of the detection surface two-dimensional coordinates, And a scanning transmission microscope apparatus.
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