JP2012167951A - Method for producing needle-like sample for electronic microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a needle-like sample for an electronic microscope which allows the needle-like sample to be simply produced.SOLUTION: The method for producing the needle-like sample to be observed by the electronic microscope includes: a step for placing a target sample on a placing sample; a step for fixing the target sample on the placing sample; a step for applying a converged ion beam to the target sample and the placing sample and producing a thin sample composed of the target sample and the placing sample; a step for soaking the thin sample in a solution and dissolving the placing sample to leave only the target sample; and a step for extracting the left target sample from the solution.

Description

  The present invention relates to a method for preparing a needle-like sample for an electron microscope, and more specifically, tomography is performed using a general-purpose transmission electron microscope (TEM) or a scanning transmission electron microscope (STEM). The present invention relates to a method for producing a needle-like sample for an electron microscope, which can easily produce a needle-like sample to be used for performing.

  In TEM and STEM tomography generally known in the industrial and academic fields, there are cases where three-dimensional observation and three-dimensional analysis of a sample are performed. A CT (Computerized Tomography) method is known as a method for irradiating an object with an X-ray, an electron beam or the like to know the internal structure of the object in a three-dimensional manner. The X-ray CT method using X-rays is widely used in the medical field, but the TEM electron beam CT method (TEM tomography or electron beam tomography) is a method for observing the three-dimensional structure of a very small region of a sample. It has been used in the fields of materials, medicine, and biology.

  In order to construct a three-dimensional image, it is necessary to obtain the relationship between the object cross section and the projected image. Therefore, in the electron beam CT method using TEM, there is a method of obtaining a transmission electron microscope image (hereinafter referred to as TEM image) of various orientations, that is, a continuous tilt image by tilting the sample.

  FIG. 5 is a diagram illustrating a configuration example of a transmission electron microscope. FIG. 5 shows a configuration example of a TEM that performs electron beam CT. In the figure, 100 is a TEM electron optical system, 1 is an electron beam source, 2 is a focusing lens group for focusing the electron beam and adjusting the amount of electron beam irradiation to the sample, and 3 is a movement of the observation position of the sample. A goniometer stage for tilting, 4 is a sample holder (sample holder) for holding the sample S, 5 is an objective lens for enlarging the electron beam transmitted through the sample, and 6 is an aperture for limiting the capture angle of the objective lens. , 7 is an objective minilens that is used in combination with or independently of the objective lens, and 8 is a stop for limiting the capture angle of the objective minilens 7.

  9 is an intermediate / projection optical system for enlarging the image surface of the objective lens 5 or the objective mini lens 7 on the detection surface, 10 is a CCD camera for TEM image observation, 11 is an acceleration voltage setting and each lens in the electron optical system 100 An electron optical system control unit capable of controlling excitation, on / off, and selection of each aperture diameter, 12 an image processing device for converting the output of the CCD camera 10 into an image, and 13 a control arithmetic device for controlling and calculating the entire TEM It is. For example, a personal computer is used as the control arithmetic device 13.

  Reference numeral 14 denotes a display device such as a liquid crystal monitor, and 15 denotes an input device such as a mouse and a keyboard. The electron beam path inside the electron optical system 100 is maintained at a high vacuum by a vacuum exhaust device (not shown).

  In the TEM configured as described above, a control signal is sent from the control arithmetic unit 13 to the goniometer stage (hereinafter simply referred to as goniometer) 3, and the transmitted electron beam image is obtained while the sample S is inclined stepwise by a predetermined angle. The obtained plurality of transmission electron beam images are converted into images by the image processing device 12, and the converted image is obtained by the control arithmetic device 13 based on the plurality of images so as to obtain a three-dimensional image. It has become. The obtained three-dimensional image is displayed on the display device 14.

  FIG. 6 is a diagram illustrating the principle of tomography creation. In this method, first, the TEM image or the STEM image 20 is acquired by inclining the sample S at various angles using TEM or STEM. For example, if the setting range of the tilt angle is tilted by 1 ° at ± 60 ° and the TEM image or STEM image is acquired simultaneously, the total number of acquired tilt images is 121. The reconstructed cross-sectional image 21 is obtained by applying the CT method to the acquired tilt image series. Here, an existing technique is used as the CT method. Then, the three-dimensional image 22 is obtained by superposing the obtained series of reconstructed sectional images.

  As a conventional apparatus of this type, there is known an apparatus that corrects a positional deviation of a sample by cutting out the same field of view by two-dimensional correlation processing with a reference image from a series of transmission images obtained by tilting the sample at a certain angle. (See, for example, Patent Document 1).

JP-A-2005-19218 (paragraphs 0023 to 0027, paragraphs 0044 to 0050, FIGS. 1 to 3)

  As described above, in order to obtain a three-dimensional image, an omnidirectional transmission image is required. However, the tilt angle is limited by the physical arrangement of the goniometer for tilting the sample and the optical system of the sample, and it is difficult to obtain a transmission image in all directions. In this way, the reconstructed cross-sectional image 21 obtained by applying the CT method from the tilt image series in which the tilt angle is limited causes the image to extend in the thickness direction and cause a reduction in spatial resolution in the sample thickness direction.

In order to solve this problem, it is necessary to cancel the limitation on the tilt angle. However, when the TEM or STEM is used, the tilt angle is limited due to two major causes.
1) Since the sample holder 4 for observing and tilting the sample S physically interferes with the pole piece of the electromagnetic lens, it cannot tilt in all directions.
2) Since the thickness at which the electron beam passes through the sample S increases as the tilt angle increases, the transmittance of the electron beam decreases and the image itself cannot be obtained.

  In recent years, the latter problem has been solved by making the sample shape into a needle shape using a focused ion beam apparatus (FIB). However, there is a problem that the preparation of the needle-shaped sample requires a very advanced technique and a lot of time.

  The present invention has been made in view of such a problem, and is capable of easily producing a needle-like sample for use in tomography using a TEM or STEM. It aims to provide a method.

  In order to solve the above-described problems, the present invention has the following configuration.

  (1) The invention according to claim 1 is a method for producing a needle-like sample for observation with an electron microscope, the step of placing a target sample on a sample for mounting, and the target sample described above A step of fixing on a mounting sample, a step of irradiating the target sample and the mounting sample with a focused ion beam to produce a thin sample comprising the target sample and the mounting sample, and the thin sample In the solution, the step of dissolving the sample for mounting described above to make only the target sample, and the step of taking out the remaining target sample from the solution.

  (2) The invention described in claim 2 is characterized in that the thin piece sample is placed on a sample holding material, and the sample holding material is placed in the solution.

  (3) The invention described in claim 3 is characterized in that the sample holding material is a grid attached to a sample holder of an electron microscope.

  The present invention has the following effects.

  (1) According to the invention described in claim 1, a needle-shaped sample can be easily produced.

  (2) According to the invention described in claim 2, only the target sample can be taken out by melting the mounting sample.

  (3) According to the invention described in claim 3, the target sample can be reliably held by using the grid as the sample holding material.

It is a figure which shows the preparation procedure of a needle-shaped sample. It is explanatory drawing of polymer sample B removal from a thin piece sample. It is a figure which shows the transmittance | permeability characteristic of a sample. It is a figure which shows the permeation | transmission state of the electron beam to a thin piece sample. It is a figure which shows the structural example of a transmission electron microscope. It is a creation principle figure of tomography.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a diagram showing a procedure for producing a needle-like sample. In the figure, the procedure consists of (a) to (l). Hereinafter, each procedure will be described in detail. In the drawing, the gray region is a region where the sample S is cut (processed) by the ion beam. In the figure, (a) to (d) are in the FIB apparatus, (e) and (f) are in the atmosphere, and (g) to (l) are in the FIB apparatus.
(A) Place the sample on the FIB sample placement section. Here, sample A is used as sample S. As the sample A, various samples such as a biological sample can be used. First, the A1 portion of the sample A is shaved with an ion beam.
(B) Since a portion A5 surrounded by two holes is formed as shown in the figure, the bottom portion A2 of A5 is shaved with an ion beam.
(C) The side surface A3 of the sample portion A5 is shaved with an ion beam to make it thin.
(D) The portion A4 where the sample portion A5 is connected to the sample A is shaved with an ion beam.
(E) The thin piece sample A5 that can be removed from the sample A is taken out by bonding with the glass probe 30 using a pickup system. At this time, the glass probe 30 adheres the thin piece sample A5 by static electricity. Thereby, the thin piece sample A5 can be easily taken out.
(F) A thin sample (target sample) A5 picked up on a bulk polymer sample (mounting sample) B is placed. Here, the sample B is not limited to the polymer sample, but may be any other material that is soluble in the solution (details will be described later).
(G) Both ends of the thin piece sample A5 placed on the polymer sample B are fixed by the carbon vapor deposition 31. Carbon vapor deposition is one of the methods for fixing the thin sample A5 to the polymer sample B, and is not necessarily carbon vapor deposition. Other fixing methods can be used.
(H) The B1 portion is shaved with an ion beam.
(I) As shown in the figure, since the portion B5 surrounded by the two holes is formed, the bottom portion B2 of B5 is shaved with an ion beam.
(J) The side surface B3 of the sample portion B5 is shaved with an ion beam to make it thin.
(K) The portion B4 where the sample portion B5 is connected to the sample B is shaved with an ion beam.
(L) The sample portion B5 is taken out using the glass probe 30 of the pickup system.

  By the above procedure, a thin sample having a thickness that can be observed by TEM is formed. At this time, the extracted thin piece sample B5 is a convex thin piece sample with the carbon vapor deposition 31 attached to both ends. In this state, since the polymer sample is attached to the sample B5, it is necessary to remove the polymer sample.

  FIG. 2 is an explanatory diagram of the removal of the polymer sample B from the thin piece sample B5. Reference numeral 34 denotes a grid attached to a sample holder of an electron microscope, on which a sample B5 with a polymer sample B (32) is placed. By using the grid 34, the thin piece sample can be stably held. The polymer sample B and the target sample A5 'are placed on the grid 34. The sample B5 placed on the grid 34 is immersed in the solution 35. The solution 35 is contained in a container such as a bottle. This solution 35 is a solution in which only the polymer sample B is dissolved. As the solution, for example, acetone is used. Thereafter, when the grid 34 is pulled up from the solution, only the target sample A5 'remains. The carbon vapor deposition 31 remains attached to both ends of the target sample A5 '. The sample thus obtained is called a needle sample. With the above method, a needle-shaped sample can be prepared very easily.

  The needle sample A5 'is very useful for tomography. The reason is as follows. FIG. 3 is a diagram showing the transmittance characteristics of the needle-shaped sample and the normal sample. The horizontal axis is the tilt angle, and the vertical axis is the transmittance. In the figure, the characteristics of three types of samples f1 to f3 are shown. The thick solid line in the figure represents the transmittance with respect to the inclination angle of the needle-shaped sample, and the thin solid line represents the transmittance with respect to the inclination angle of the normal sample. Here, the transmittance indicates a ratio of transmitting through the sample. When the transmittance is 1.0, it indicates that all the beams are transmitted, and when 0, the transmittance is not transmitted at all. However, the needle-like sample is assumed to be rectangular. FIG. 4 is a diagram showing a transmission state of the electron beam to the thin piece sample. The θ in the figure is the sample tilt angle. In the case of the figure, the sample inclination angle is 45 degrees. In the case of a needle sample, the transmittance is the worst when the sample tilt angle is 45 degrees (described later). The inclination angle θ of the needle-like sample is set by the goniometer 3, and a transmission image is obtained while being changed by a predetermined angle.

  In the case of a normal sample, the transmittance decreases as the tilt angle increases. In particular, even with a normal sample having a transmittance of 0.6 and an inclination angle of 0 °, the transmittance is almost 0 when the inclination angle is 80 °. In this case, an image cannot be obtained because it cannot be transmitted even if it can be physically tilted. On the other hand, in the case of a needle-like sample, it can be seen that the transmittance is lowest when the tilt angle is 45 degrees, and the transmittance increases when the tilt angle is more than that. This is because when a quadrangular prism is used as the needle-shaped sample, the sample thickness becomes the largest when the inclination angle is 45 degrees. Also, the difference between the lowest transmittance and the highest transmittance is about 0.1 degree, and an image with a high transmittance can be obtained at all angles. The thin sample prepared as described above can be observed as a three-dimensional image with a transmission electron microscope shown in FIG.

  That is, after setting the needle-like sample A5 ′ produced by the above-described method to the sample holder 4, the transmission image 20 is obtained by changing the inclination by a predetermined angle with the goniometer 3, and the reconstructed cross-sectional image 21 is obtained from the obtained transmission image 20. Get. A three-dimensional image 22 can be obtained by superimposing the obtained reconstruction sectional images 21.

  In addition, although the case where the thing of a quadrangular prism shape was used as an example of the shape of a needle-shaped sample was taken as an example, this invention is not limited to this, For example, a triangular prism shape or a pentagonal prism shape may be used.

  As described above in detail, according to the present invention, a needle-like sample used for tomography using a TEM or STEM can be easily produced, and a practical effect is great.

30 Glass probe 31 Carbon deposition A5 Thin sample A5 'Thin sample B Polymer sample

Claims (3)

A method of producing a needle-like sample for observation with an electron microscope, the step of placing a target sample on a sample for placement;
Fixing the target sample on the mounting sample;
Irradiating the target sample and the mounting sample with a focused ion beam to produce a thin sample comprising the target sample and the mounting sample;
Placing the thin piece sample in a solution, dissolving the sample for mounting as described above to make only the target sample; and
And a step of taking out the remaining target sample from the solution.   2. The method for producing a needle-shaped sample for an electron microscope according to claim 1, wherein the thin sample is placed on a sample holding material, and the sample holding material is placed in the solution.   The method for producing a needle-shaped sample for an electron microscope according to claim 2, wherein the sample holding material is a grid attached to a sample holder of an electron microscope.

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