JP7586779B2 - Sample observation method and cartridge - Google Patents

Description

The present invention relates to a sample observation method and a cartridge.

Biological samples with a high water content cannot be observed as is in a transmission electron microscope (TEM), so they must be frozen before observation, for example. In this way, cryo-electron microscopy, in which frozen samples are introduced into an electron microscope for observation without staining, etc., is attracting attention.

For example, Patent Document 1 discloses a cryo-electron microscope equipped with a transport device that can automatically transport a cartridge with a cooled sample fixed thereto from the sample exchange chamber to the sample chamber.

JP 2015-088237 A

To observe a sample using this type of cryo-electron microscopy, for example, the sample is first cooled and frozen in a cooling device, and then sliced into thin slices using a processing device. The sliced sample is then introduced into a transmission electron microscope for observation.

In this way, in order to observe a sample, it must be processed using multiple processing equipment from the time the sample is frozen until it is observed. Because each processing equipment has a different stage and holder, it is necessary to transfer the sample from the time the sample is frozen until it is observed. When transferring the sample, the worker must use tweezers or the like to handle the TEM grid that supports the sample. Sample supports such as TEM grids are thin and small, making this work very difficult. Furthermore, there is a risk that the sample will fall off or be damaged during this type of work.

One aspect of the sample observation method according to the present invention includes:
A sample observation method in which a sample is frozen and observed under a transmission electron microscope, comprising the steps of:
supporting the frozen sample in a cartridge;
Observing the sample supported by the cartridge with an optical microscope and acquiring position information of an observation target;
introducing the cartridge into a focused ion beam device and processing the sample based on the position information;
introducing the cartridge into the transmission electron microscope and observing the processed sample with the transmission electron microscope;
Includes.

In this type of sample observation method, the sample is supported in a cartridge from the time the sample is observed with an optical microscope until the processed sample is observed with a transmission electron microscope. This means that the operator does not need to directly handle the sample or the sample support, making it easy to prepare samples for transmission electron microscopes. Furthermore, the prepared sample can be easily introduced into the transmission electron microscope.

One aspect of the cartridge according to the present invention is
With the base,
a mounting portion provided on the base to which a sample support for an electron microscope is attached;
Including,
The base has a notch for irradiating a sample with a focused ion beam in a focused ion beam device, and a through hole for passing an electron beam in a transmission electron microscope.
was established,
The cutout is provided with a beam portion spanning the cutout.

In such a cartridge, the sample can be supported from when it is observed with an optical microscope until the processed sample is observed with a transmission electron microscope. Furthermore, in such a cartridge, a beam portion is provided in the cutout, which improves the strength of the cartridge.

FIG. 2 is a perspective view illustrating a cartridge used in the sample observation method according to the first embodiment. FIG. 2 is a cross-sectional view showing a schematic diagram of a cooled sample holder for a TEM used in the sample observation method according to the first embodiment. 4 is a flowchart showing an example of a sample observation method according to the first embodiment. FIG. 1 is a diagram for explaining a process of freezing a sample. FIG. 1 is a diagram for explaining a process of freezing a sample. FIG. 11 is a perspective view showing a process of supporting a sample with a cartridge. FIG. 1 is a perspective view illustrating a focused ion beam device. FIG. 1 is a perspective view showing a sample being processed by a focused ion beam device. FIG. 1 is a perspective view showing a sample being processed by a focused ion beam device. FIG. 1 is a schematic diagram showing a transmission electron microscope. FIG. 2 is a schematic diagram showing a sample supported by a cartridge. FIG. 11 is a diagram illustrating a modified example of the cartridge. FIG. 11 is a perspective view illustrating a cartridge used in a sample observation method according to a second embodiment. 10 is a flowchart showing an example of a sample observation method according to the second embodiment. FIG. 11 is a perspective view showing a process of supporting a sample with a cartridge. A schematic diagram showing how a sample is processed using a focused ion beam. FIG. 1 is a schematic diagram of a sliced sample.

Preferred embodiments of the present invention will be described in detail below with reference to the drawings. Note that the embodiments described below do not unduly limit the contents of the present invention described in the claims. Also, not all of the configurations described below are necessarily essential components of the present invention.

1. First embodiment 1.1. Cartridge First, a cartridge used in the sample observation method according to the first embodiment will be described with reference to the drawings. Fig. 1 is a perspective view showing a cartridge 100 used in the sample observation method according to the first embodiment.

As shown in FIG. 1, the cartridge 100 includes a base 110, a fixing groove 120, a first mounting portion 130, and a second mounting portion 140.

The base 110 is a plate-shaped member. The base 110 is provided with a fixing groove 120, a first mounting portion 130, and a second mounting portion 140.

The fixing groove 120 is a groove for mounting the cartridge 100 to a cooled sample holder 200 for a TEM shown in FIG. 2, which will be described later. The cooled sample holder 200 will be described later.

The first mounting part 130 is configured to be able to mount the frozen sample support to the cartridge 100. The frozen sample support is mounted to the base 110 by the first mounting part 130. The first mounting part 130 includes, for example, a recess provided in the base 110, and the frozen sample support is mounted to the base 110 by fitting the frozen sample support into the recess. The frozen sample support is a support for supporting a sample when the sample is frozen in a freezing device. The frozen sample support is, for example, a freezing dish.

The second mounting portion 140 is configured to be able to mount the TEM sample support to the cartridge 100. The TEM sample support is mounted to the base 110 by the second mounting portion 140. The second mounting portion 140 includes, for example, a recess provided in the base 110, and the TEM sample support is mounted to the base 110 by fitting the TEM sample support into the recess. The TEM sample support is a support for supporting a sample for a TEM. The TEM sample support is, for example, a TEM grid, a notched grid that can be used in a focused ion beam device, etc.

When a TEM sample support is attached to the second mounting portion 140, the sample supported by the TEM sample support is located, for example, at the center of the base 110 in the thickness direction of the base 110. In other words, when a TEM sample support is attached to the second mounting portion 140, the distance between the sample and the upper surface 110a of the base 110 is equal to the distance between the sample and the lower surface 110b of the base 110.

As shown in FIG. 1, the cartridge 100 has a notch 150 for irradiating a sample with a focused ion beam in a focused ion beam device, and a through hole 160 for passing an electron beam in a transmission electron microscope.

The notch 150 is formed by cutting out the side surface 102 of the base 110. The notch 150 is connected to a recess in the second mounting portion 140. Therefore, a focused ion beam can be irradiated through the notch 150 to a sample supported by a TEM sample support attached to the second mounting portion 140.

The base 110 has a first surface 111, a second surface 112, a third surface 113, and a fourth surface 114 that define the notch 150. The first surface 111 and the fourth surface 114 are connected to the side surface 102 of the base 110. The second surface 112 connects the first surface 111 to the surface that defines the second mounting portion 140. The third surface 113 connects the fourth surface 114 to the surface that defines the second mounting portion 140.

The cutout 150 is provided with a beam portion 170 that straddles the cutout 150. The beam portion 170 connects two of the multiple surfaces that define the cutout 150, sandwiching the cutout 150. The first surface 111 and the fourth surface 114 are the two surfaces that sandwich the cutout 150, and the beam portion 170 connects the first surface 111 and the fourth surface 114.

By providing the beam portion 170 in the cutout 150, the strength of the cartridge 100 can be improved compared to, for example, a case in which the beam portion 170 is not provided in the cutout 150.

The through-hole 160 is provided in the bottom surface of the recess of the second mounting part 140. In a transmission electron microscope, when an electron beam is irradiated onto a sample supported by a TEM sample support attached to the second mounting part 140, the electron beam that passes through the sample passes through the through-hole 160.

1.2 Cooled Sample Holder Next, the cooled sample holder for a TEM used in the sample observation method according to the first embodiment will be described with reference to the drawings. Fig. 2 is a cross-sectional view showing a schematic diagram of a cooled sample holder 200 for a TEM used in the sample observation method according to the first embodiment.

The cooled sample holder 200 is a sample holder that can cool a frozen sample. By using the cooled sample holder 200, a frozen sample can be held without melting. As shown in FIG. 2, the cooled sample holder 200 includes a cartridge holding section 210, a heat conductor 220, a tank 230, and a cover 240.

The cartridge holding section 210 is provided at the tip of the cooling sample holder 200. The cartridge holding section 210 can hold the cartridge 100. The cartridge holding section 210 holds the cartridge 100, for example, by inserting a fastener (not shown) into the fixing groove 120 of the cartridge 100.

The tank 230 contains a cooling medium. For example, liquid nitrogen can be used as the cooling medium. The thermal conductor 220 is made of a material with high thermal conductivity. One end of the thermal conductor 220 is connected to the cooling medium of the tank 230, and the other end of the thermal conductor 220 is connected to the cartridge holder 210. Therefore, the cartridge 100 can be cooled.

The cover 240 covers the cartridge 100 held in the cartridge holding section 210. By covering the cartridge 100 with the cover 240, it is possible to prevent frost from adhering to the cartridge 100 or the sample supported by the cartridge 100.

1.3 Sample Observation Method Next, a sample observation method according to the first embodiment will be described. Fig. 3 is a flowchart showing an example of the sample observation method according to the first embodiment.

1.3.1. Freezing the sample S100
First, a sample is frozen. The sample is, for example, a biological sample such as a cell, a protein, a virus, a lipid molecule, etc. By freezing a biological sample with a high water content, it can be observed with a transmission electron microscope without staining or the like.

The freezing of the sample is carried out using a freezing device. For example, a high-pressure freezing device can be used as the freezing device. In the high-pressure freezing device, the sample is frozen by applying pressure, and the water can be frozen amorphously deeper into the sample than when it is frozen under atmospheric pressure.

Figures 4 and 5 are diagrams for explaining the process of freezing sample 2.

As shown in FIG. 4, first, the sample 2 is sandwiched between two frozen sample supports 10. Next, the two frozen sample supports 10 sandwiching the sample 2 are set in a high-pressure freezing device. Next, the sample 2 is frozen in the high-pressure freezing device. Next, the sample 2 sandwiched between the two frozen sample supports 10 is fractured in a cooling fracture device. In this way, as shown in FIG. 5, the sample 2 supported by the frozen sample supports 10 can be obtained. By fracturing the sample 2 in the cooling fracture device, a flat surface can be formed on the sample 2. This makes it easier to process the sample 2 using a focused ion beam.

In the cryo-fracturing apparatus, the sample 2 is placed in a vacuum or nitrogen gas atmosphere to prevent frost from forming on the sample 2. The sample 2 supported on the frozen sample support 10 shown in Figure 5 is placed in a liquid nitrogen cup and transported to the workstation.

1.3.2. Step S102 of supporting the sample in a cartridge
FIG. 6 is a perspective view that typically illustrates step S102 of supporting the sample 2 with the cartridge 100.

As shown in FIG. 6, the sample 2 supported on the frozen sample support 10 is attached to the cartridge 100. In the workstation, the frozen sample support 10 is attached to the first attachment portion 130. The workspace of the workstation is filled with liquid nitrogen, so that the frozen sample support 10 can be attached to the cartridge 100 without frosting the sample 2.

Before attaching the frozen sample support 10 to the cartridge 100, the TEM sample support 20 is attached to the second attachment portion 140 of the cartridge 100.

In this way, in this process, both the frozen sample 2 and the TEM sample support 20 are mounted in the cartridge 100. Therefore, in the subsequent processes, the operator only needs to handle the cartridge 100, and does not need to directly handle the sample 2 or the TEM sample support 20.

1.3.3. Step S104 of observing the sample with an optical microscope
The cartridge 100 supporting the frozen sample 2 and the TEM sample support 20 is transported to the optical microscope, and the cartridge 100 is attached to the stage of the optical microscope. Note that the cartridge 100 is transported from the workstation to the optical microscope using a liquid nitrogen cup.

The stage of the optical microscope can carry the cartridge 100. The stage of the optical microscope is connected to a liquid nitrogen tank, and can cool the cartridge 100. The cartridge 100 carried on the stage is placed in, for example, a pressurized nitrogen gas atmosphere. This allows the sample 2 to be observed with the optical microscope without frost forming on the sample 2.

In the optical microscope, the sample 2 is observed, and position information of the observation target is obtained. The position of the observation target can be specified, for example, by a plurality of alignment markers provided on the cartridge 100. Specifically, in the optical microscope, the stage is moved to the position of each alignment marker, and the stage coordinates of each alignment marker are recorded. Next, the stage is moved to the position of the observation target, and the stage coordinates of the observation target are recorded. In this manner, position information of the observation target is obtained. The position information of the observation target includes, for example, the stage coordinates of each alignment marker and the stage coordinates of the observation target. The alignment markers may be provided on the TEM sample support 20.

For example, a fluorescence microscope may be used as an optical microscope for observing the sample 2. With the fluorescence microscope, the fluorescence emitted from the sample 2 can be observed.

1.3.4. Step S106: Attaching the Cartridge to the Cooling Sample Holder
After obtaining position information of the observation target with the optical microscope, the cartridge 100 is transported from the optical microscope to the workstation using a liquid nitrogen cup. In the workstation, the cartridge 100 is attached to the cooled sample holder 200 shown in FIG. 2. Specifically, in the workstation, the cartridge 100 is attached to the cartridge holding portion 210 of the cooled sample holder 200 in liquid nitrogen. After the cartridge 100 is attached to the cartridge holding portion 210, the cartridge 100 is covered with a cover 240. Since the cartridge holding portion 210 is thermally connected to the tank 230, the cartridge 100 held by the cooled sample holder 200 is cooled.

1.3.5. Step S108 of processing the sample into a plate shape using a focused ion beam device
The cartridge 100 is transported to a processing chamber of a focused ion beam device using the cooled sample holder 200. Then, the sample 2 is processed using the focused ion beam device.

Figure 7 is a perspective view showing a schematic diagram of a focused ion beam device 300. As shown in Figure 7, the focused ion beam device 300 includes an electron beam column 310, a gas injection system 320, a focused ion beam column 330, a manipulator system 340, a processing chamber 350, a transport rod 360, and a sample chamber 370.

The focused ion beam device 300 is equipped with an electron beam column 310 and a focused ion beam column 330, and is capable of processing the sample 2 using a focused ion beam and observing the sample 2 using a scanning electron microscope.

As shown in FIG. 7, the processing of sample 2 begins by inserting the cooled sample holder 200 into the processing chamber 350 and attaching the cartridge 100 to the table inside the processing chamber 350. After the cartridge 100 is attached to the table, the cooled sample holder 200 is removed. The processing chamber 350 is kept in a vacuum state, and the table is connected to a liquid nitrogen tank, so that the sample 2 can be kept frozen even inside the processing chamber 350.

In the processing chamber 350, a conductive coating is applied to the frozen sample 2. The processing chamber 350 is maintained in a vacuum state by an exhaust system independent of the sample chamber 370. Therefore, argon gas can be flowed only in the processing chamber 350 to perform sputter coating on the sample 2.

After coating the sample 2, the cartridge 100 is transported from the processing chamber 350 to the sample chamber 370 using the transport rod 360. The cartridge 100 transported to the sample chamber 370 is fixed to a cooling stage arranged within the sample chamber 370.

Figures 8 and 9 are perspective views that show the process of processing a sample 2 using a focused ion beam device 300.

8, the sample 2 is processed based on the position information of the observation target. For example, the alignment markers provided on the cartridge 100 are found by observing the cartridge 100 with a scanning electron microscope (electron beam column 310), and the stage coordinates of each alignment marker are recorded. The recorded stage coordinates are compared with the position information of the observation target (position information of each alignment marker), and the stage coordinates of the observation target in the focused ion beam device 300 are obtained.

In the above, we have described a case where the alignment marker is checked using an SEM image that uses an electron beam when aligning the observation object, but the alignment marker may also be checked using a SIM (Scanning Ion Microscope) image that uses an ion beam.

Once the stage coordinates of the observation target have been identified, the sample 2 is moved to the identified stage coordinates. Then, the focused ion beam is irradiated onto the sample 2 from the focused ion beam column 330, and the sample 2 is processed.

When processing sample 2, first, a deposition film is formed on sample 2 as a protective film. Specifically, the nozzle of the gas injection system 320 is placed near sample 2, and Pt organic compound gas is sprayed onto sample 2. This allows a deposition film to be formed on the surface of sample 2. By forming a protective film on the surface of sample 2, damage to the object being observed during processing can be reduced.

Since it is not possible to form a film by CVD (Chemical Vapor Deposition) using an electron beam or ion beam at low temperatures, the film is formed by spraying gas onto the low-temperature sample 2 as described above.

Next, the sample 2 is processed using a focused ion beam. Specifically, first, a part of the sample 2 is processed into a plate shape using a focused ion beam. Next, the bottom and sides of the plate-shaped part are cut off using a focused ion beam. At this time, the entire side is not cut off, and the plate-shaped part is left connected to the remaining part of the sample 2.

1.3.6. Step S110 of transferring the sample to a TEM sample support by a manipulator
Next, the manipulator system 340 is used to transfer the plate-shaped sample 2 from the frozen sample support 10 to the TEM sample support 20 .

Specifically, first, the tip of the manipulator 342 is fixed to the plate-shaped portion of the sample 2, and the manipulator 342 is connected to the plate-shaped portion. Next, a focused ion beam is used to cut off the portion where the plate-shaped portion and the remaining portion of the sample 2 are connected, and the plate-shaped portion is separated. Next, as shown in FIG. 9, the manipulator 342 transports the plate-shaped portion from the frozen sample support 10 to the TEM sample support 20. Next, the plate-shaped portion is connected to the TEM sample support 20, and the connection portion between the manipulator 342 and the plate-shaped portion is cut off. Through the above steps, the plate-shaped sample 2 can be transferred to the TEM sample support 20.

1.3.7. Step S112 of thinning the sample using a focused ion beam device
Next, the sample 2 supported on the TEM sample support 20 is sliced by a focused ion beam. By sliced, the sample 2 can be observed by a transmission electron microscope.

After the sample 2 has been thinned, the cartridge 100 is transported from the sample chamber 370 to the processing chamber 350 by the transport rod 360. The cooled sample holder 200 is inserted into the processing chamber 350, and the cartridge 100 is removed from the processing chamber 350.

In addition, the sliced sample 2 may be coated with a conductive film in the processing chamber 350. This can reduce sample drift caused by charging when observing the sample 2 with a transmission electron microscope.

1.3.8. Observing the sample with a transmission electron microscope (S114)
In the workstation, the cartridge 100 is removed from the cooled specimen holder 200, and the removed cartridge 100 is loaded into a magazine. The magazine loaded with the cartridge 100 is attached to the transmission electron microscope. This allows the cartridge 100 to be introduced into the transmission electron microscope. As a result, the observation target of the specimen 2 can be observed with the transmission electron microscope.

Figure 10 is a schematic diagram of a transmission electron microscope 400.

The transmission electron microscope 400 forms a TEM image using an electron beam transmitted through the sample 2. The transmission electron microscope 400 is a cryo-electron microscope that can observe the sample 2 while cooling it. As shown in FIG. 10, the transmission electron microscope 400 includes a gate valve 420, a transport device 430, a transport device 440, a gate valve 450, and a sample holder 460.

The gate valve 420 is a valve that separates the sample exchange chamber 404 and the liquid nitrogen cup 410. The liquid nitrogen cup 410 is attached to the sample exchange chamber 404 via the gate valve 420. The transport device 430 transports the magazine 412 between the liquid nitrogen cup 410 and the sample exchange chamber 404. The magazine 412 can hold a plurality of cartridges 100. The transport device 440 transports the cartridges 100 between the sample exchange chamber 404 and the sample chamber 402. The gate valve 450 is a valve that separates the sample exchange chamber 404 and the sample chamber 402. The sample holder 460 can hold the sample 2 while cooling it.

When introducing the cartridge 100 into the sample chamber 402 in the transmission electron microscope 400, first, the liquid nitrogen cup 410 that contains the magazine 412 to which the cartridge 100 is attached is attached to the sample exchange chamber 404 via the gate valve 420. After opening the gate valve 420, the transport device 430 grasps the magazine 412 and introduces the magazine 412 into the sample exchange chamber 404. In the sample exchange chamber 404, the transport device 440 grasps the cartridge 100 attached to the magazine 412. The gate valve 450 is opened, and the transport device 440 transports the cartridge 100 from the sample exchange chamber 404 to the sample chamber 402. In the sample chamber 402, the cartridge 100 is handed over to the sample holder 460. This allows the cartridge 100, i.e., the sample 2, to be introduced into the sample chamber 402.

In the sample chamber 402, the sample 2 is irradiated with an electron beam. This allows a TEM image to be obtained.

As described above, in the transmission electron microscope 400, the cartridge 100 is transported by the transport device 440. When the transport device 440 stores the cartridge 100 in the magazine 412, or when the cartridge 100 is transferred between the transport device 440 and the sample holder 460, a force is applied to the cartridge 100.

For example, if the cutout 150 of the cartridge 100 does not have a beam 170, the strength of the portion of the cartridge 100 where the cutout 150 is provided is low, and the cartridge 100 may become deformed. In contrast, the cartridge 100 has a beam 170 in the cutout 150, so the strength of the cartridge 100 is high and deformation of the cartridge 100 can be prevented.

The transmission electron microscope may be configured, for example, so that the cooled sample holder 200 can be directly inserted. This makes it easy to introduce the sliced sample 2 into the transmission electron microscope.

Figure 11 is a schematic diagram showing a sample 2 supported by a cartridge 100.

As shown in FIG. 11, the sample 2 is supported by a TEM sample support 20. The TEM sample support 20 is attached to a second mounting portion 140. Here, the cartridge 100 has a notch 150, so that the sample 2 can be processed with a focused ion beam. Furthermore, the cartridge 100 has a through hole 160, so that the sample 2 can be observed in a transmission electron microscope.

1.4. Effects The sample observation method according to the first embodiment includes a step of supporting the frozen sample 2 in the cartridge 100, a step of observing the sample 2 supported by the cartridge 100 with an optical microscope and acquiring position information of the observation target, a step of introducing the cartridge 100 into the focused ion beam device 300 and processing the sample 2 based on the position information of the observation target, and a step of introducing the cartridge 100 into a transmission electron microscope and observing the processed sample 2 with the transmission electron microscope. Thus, in the sample observation method according to the first embodiment, the sample 2 is supported by the cartridge 100 from the observation of the sample 2 with the optical microscope until the observation of the processed sample 2 with the transmission electron microscope. Therefore, the operator does not need to directly handle the sample 2 or the sample support, and can easily prepare a sample for a transmission electron microscope. Furthermore, the prepared sample can be easily introduced into the transmission electron microscope.

For example, if the cartridge 100 was not used, the operator would have to place the grid on which the sample 2 was supported on the FIB sample holder, and then transfer the grid from the FIB sample holder to the TEM sample holder after processing was completed.

The sample observation method according to the first embodiment eliminates the need for such work, making it easy to prepare samples for transmission electron microscopes.

The sample observation method according to the first embodiment includes a step of freezing the sample 2 supported on the frozen sample support 10 in a freezing device, and in the step of supporting the frozen sample 2 in the cartridge 100, the frozen sample support 10 is attached to the first attachment portion 130 of the cartridge 100, and in the step of processing the sample 2, the sample 2 supported on the frozen sample support 10 is processed based on position information of the observation target, the processed sample 2 is transferred to the TEM sample support 20 attached to the second attachment portion 140 of the cartridge 100, and the sample 2 supported on the TEM sample support 20 is processed to slice the sample 2.

In this way, in the sample observation method according to the first embodiment, after the sample 2 is frozen in the freezing device, the frozen sample 2 is supported by the cartridge 100 until it is observed by a transmission electron microscope. Therefore, the operator does not need to directly handle a sample support such as a TEM grid.

In addition, both the frozen sample support 10 and the TEM sample support 20 are pre-attached to the cartridge 100. This makes it easy to share coordinates between the processing devices.

For example, if the cartridge 100 was not used, when transferring a grid from an FIB sample holder to a TEM sample holder, it was necessary to share coordinates taking into account the rotation of the grid. This is because grids are generally circular, and when the grid is attached to a TEM sample holder, the grid has a degree of freedom of rotation.

In contrast, when the cartridge 100 is used, the TEM sample support 20 is fixed to the cartridge 100, so there is no need to consider grid rotation when sharing coordinates. In addition, since both the frozen sample support 10 and the TEM sample support 20 are attached to the cartridge 100 in advance, a common alignment marker can be used. Therefore, it is easy to share coordinates.

In the sample observation method according to the first embodiment, in the process of processing the sample 2, the processed sample 2 is transferred to the TEM sample support 20 using a manipulator 342 mounted on the focused ion beam device 300. Therefore, the sample 2 can be transferred from the frozen sample support 10 to the TEM sample support 20 more reliably.

In the sample observation method according to the first embodiment, the cartridge 100 is provided with a notch 150 for irradiating the sample 2 with a focused ion beam in the focused ion beam device 300 and a through hole 160 for passing an electron beam in a transmission electron microscope, and the notch 150 is provided with a beam portion 170 that straddles the notch 150. This improves the strength of the cartridge 100.

In addition, by providing the beam portion 170, the increase in temperature of the sample 2 due to radiant heat can be reduced compared to when the beam portion 170 is not provided.

The base 110 of the cartridge 100 has a first surface 111 and a fourth surface 114 that define the notch 150, the first surface 111 and the fourth surface 114 sandwich the notch 150, and the beam portion 170 connects the first surface 111 and the fourth surface 114. This improves the strength of the cartridge 100.

In the sample observation method according to the first embodiment, the transmission electron microscope includes a sample chamber 402, a sample exchange chamber 404 connected to the sample chamber 402 via a gate valve 450, and a transport device 440 that transports the cartridge 100 between the sample exchange chamber 404 and the sample chamber 402. The cartridge 100 is provided with a beam portion 170 that straddles the notch 150, so that deformation of the cartridge 100 can be prevented even when the cartridge 100 is transported by the transport device 440.

The cartridge 100 includes a base 110 and a second mounting portion 140 that is provided on the base 110 and to which a TEM sample support 20 is attached. The base 110 is also provided with a notch 150 for irradiating a focused ion beam onto a sample 2 in a focused ion beam device 300 and a through hole 160 for passing an electron beam in a transmission electron microscope 400, and the notch 150 is provided with a beam portion 170 that straddles the notch 150.

By using the cartridge 100, the sample can be supported from when it is observed with an optical microscope until the processed sample is observed with a transmission electron microscope. Furthermore, in the cartridge 100, the beam portion 170 is provided in the cutout 150, so that the strength of the cartridge 100 can be improved. This makes it possible to prevent deformation of the cartridge 100, for example, even when the cartridge 100 is transported by the transport device 440 in a transmission electron microscope.

12 is a diagram showing a schematic diagram of a modification of the cartridge 100. Fig. 12 corresponds to Fig. 11 .

As shown in Fig. 12, the cartridge 100 does not need to have the notch 150. In the cartridge 100 shown in Fig. 12, the second mounting portion 140 supports the TEM sample support 20 at an angle relative to the upper surface 110a of the base 110. This means that the base 110 does not block the focused ion beam even if the notch 150 is not provided. Therefore, in the cartridge 100, the sample 2 can be processed with the focused ion beam, and the processed sample 2 can be observed with a transmission electron microscope.

The cartridge 100 shown in FIG. 12 can achieve the same effects as the cartridge 100 shown in FIG. 1 and FIG. 11 described above.

2. Second embodiment 2.1 Cartridge Next, a sample observation method according to the second embodiment will be described. First, a cartridge used in the sample observation method according to the second embodiment will be described with reference to the drawings.

Figure 13 is a perspective view showing a cartridge 500 used in the sample observation method according to the second embodiment. In the cartridge 500 shown in Figure 13, components having the same functions as the components of the cartridge 100 shown in Figure 1 described above are given the same reference numerals, and detailed descriptions thereof will be omitted.

As shown in FIG. 13, the cartridge 500 does not have an attachment portion (first attachment portion 130) for attaching the frozen sample support 10.

14 is a flowchart showing an example of a sample observation method according to the second embodiment. Below, the sample observation method according to the second embodiment will be described in terms of differences from the example of the sample observation method according to the first embodiment, and a description of similarities will be omitted.

2.2.1. Freezing the sample S200
In the sample observation method according to the second embodiment, a sample dropped onto a grid (one example of a sample support for TEM) or a sample cultured on the grid is frozen. For example, the sample is dropped onto the grid and then immersed together with the grid in liquid ethane or the like to freeze the sample. The frozen sample is transported to a workstation in a liquid nitrogen cup.

2.2.2. Step S202 of supporting the sample in a cartridge
FIG. 15 is a perspective view that typically illustrates step S202 of supporting the sample 2 with the cartridge 500.

In the workstation, the grid 22 holding the frozen sample 2 is attached to the second attachment part 140 (hereinafter, simply referred to as the "attachment part 140"). As in the first embodiment, in the subsequent steps, only the cartridge 500 needs to be handled, and the operator does not need to directly handle the sample 2 or the grid 22.

2.2.3. Observing the sample with an optical microscope S204
The cartridge 500 is attached to the stage of an optical microscope, and the sample 2 is observed with the optical microscope to obtain position information of the observation target. This step S204 is performed in the same manner as step S104 of observing the sample 2 with the optical microscope shown in FIG.

2.2.4. Step S206: Attaching the Cartridge to the Cooling Sample Holder
The cartridge 500 is attached to the cooled sample holder 200. This step S206 is performed in the same manner as step S106 of attaching the cartridge 100 to the cooled sample holder 200 shown in FIG.

2.2.5. Step S208 of thinning the sample using a focused ion beam device
FIG. 16 is a schematic diagram showing how a sample 2 is processed by a focused ion beam.

As shown in FIG. 16, a focused ion beam FIB is irradiated onto the sample 2 on the grid 22 to thin the sample 2. For example, the focused ion beam FIB is incident on the top surface of the grid 22 at an angle of about 10° to perform processing.

In this way, in the second embodiment, the sample 2 is frozen on the grid 22 and then processed on the grid 22, so there is no need to perform step S110 of transferring the sample 2 to a TEM sample support with a manipulator.

2.2.6. Observing the sample with a transmission electron microscope S210
The cartridge 100 is introduced into the transmission electron microscope, and the observation target of the sample 2 is observed by the transmission electron microscope. This step is performed in the same manner as step S114 of observing the sample 2 by the transmission electron microscope 400 shown in FIG.

Figure 17 is a schematic diagram of a sliced sample 2.

As shown in FIG. 17, the sample 2 includes a thin piece 2a and a mass 2b that holds the thin piece 2a. In step S208, the sample 2 is processed to form the thin piece 2a and the masses 2b located on both sides of the thin piece 2a.

When observing the sample 2 with a transmission electron microscope, it is desirable to mount the cartridge 500 on the sample stage of the transmission electron microscope so that the tilt axis L of the sample stage passes through the two masses 2b and the center of the slice 2a. This makes it possible to prevent the slice 2a from being cast in the shadow of the mass 2b and restricting the field of view when the sample 2 is tilted around the tilt axis L.

In the sample observation method according to the second embodiment, the sample 2 can be processed by the focused ion beam device 300 and observed by a transmission electron microscope while the grid 22 is supported by the cartridge 500. This makes it easy to match the orientation of the sample 2 when processed by the focused ion beam device 300 with the orientation of the sample 2 when observed by the transmission electron microscope. This prevents the tilt axis L from shifting relative to the flake 2a, resulting in the field of view being restricted by the mass 2b.

2.3 Effects The sample observation method according to the second embodiment can achieve the same effects as the sample observation method according to the first embodiment described above.

The sample observation method according to the second embodiment includes a step of freezing the sample 2 supported by the grid 22, and in the step of supporting the frozen sample 2 in the cartridge 500, the grid 22 is attached to the mounting portion 140 of the cartridge 500. This eliminates the need for a step of transferring the sample 2 from the frozen sample support 10 to the TEM sample support 20, making it easier to prepare the sample.

The cartridge 500 includes a base 110 and a mounting portion 140 that is provided on the base 110 and to which the grid 22 is attached, and the mounting portion 140 supports the grid 22 at an angle relative to the upper surface 110a of the base 110. Therefore, the strength of the cartridge 500 can be improved compared to, for example, a case in which a notch is provided in the base 110.

The present invention is not limited to the above-described embodiment, and various modifications are possible. For example, the present invention includes a configuration that is substantially the same as the configuration described in the embodiment. A substantially identical configuration is, for example, a configuration that has the same function, method, and result, or a configuration that has the same purpose and effect. The present invention also includes a configuration in which non-essential parts of the configuration described in the embodiment are replaced. The present invention also includes a configuration that has the same action and effect as the configuration described in the embodiment, or a configuration that can achieve the same purpose. The present invention also includes a configuration in which publicly known technology is added to the configuration described in the embodiment.

2...sample, 2a...thin slice, 2b...lump, 10...frozen sample support, 20...TEM sample support, 22...grid, 100...cartridge, 102...side, 110...base, 110a...top, 111...first surface, 112...second surface, 113...third surface, 114...fourth surface, 120...fixing groove, 130...first mounting portion, 140...second mounting portion, 150...notch, 160...through hole, 170...beam portion, 200...cooled sample holder, 210...cartridge holding portion, 220...thermal conductor, 230...tank, 240...cover, 300...Focused ion beam device, 310...Electron beam column, 320...Gas injection system, 330...Focused ion beam column, 340...Manipulator system, 342...Manipulator, 350...Processing chamber, 360...Transport rod, 370...Sample chamber, 400...Transmission electron microscope, 402...Sample chamber, 404...Sample exchange chamber, 410...Liquid nitrogen cup, 412...Magazine, 420...Gate valve, 430...Transport device, 440...Transport device, 450...Gate valve, 460...Sample holder, 500...Cartridge

Claims (9)

A sample observation method in which a sample is frozen and observed under a transmission electron microscope, comprising the steps of:
supporting the frozen sample in a cartridge;
Observing the sample supported by the cartridge with an optical microscope and acquiring position information of an observation target;
introducing the cartridge into a focused ion beam device and processing the sample based on the position information;
introducing the cartridge into the transmission electron microscope and observing the processed sample with the transmission electron microscope;
A sample observation method comprising: In claim 1,
freezing the sample supported on the frozen sample support in a freezing device;
In the step of supporting the frozen sample in the cartridge, the frozen sample support is attached to a first attachment portion of the cartridge;
In the step of processing the sample,
processing the sample supported on the frozen sample support based on the position information;
The processed sample is transferred to a sample support for an electron microscope attached to a second mounting portion of the cartridge;
A sample observation method, comprising processing the sample supported on the electron microscope sample support to slice the sample. In claim 2,
A sample observation method, wherein in the step of processing the sample, the processed sample is transferred to the electron microscope sample support using a manipulator mounted on the focused ion beam device. In claim 1,
freezing the sample supported on an electron microscope sample support;
A sample observation method, wherein in the step of supporting the frozen sample in the cartridge, the sample support for an electron microscope is attached to an attachment portion of the cartridge. In any one of claims 2 to 4,
the cartridge is provided with a notch for irradiating the sample with a focused ion beam in the focused ion beam device, and a through hole for passing an electron beam in the transmission electron microscope;
A sample observation method, wherein the notch is provided with a beam portion spanning the notch. In claim 5,
The cartridge includes a base;
the base has two surfaces that define the notch;
The two surfaces sandwich the notch,
A sample observation method, wherein the beam portion connects the two surfaces. In any one of claims 2 to 4,
The cartridge includes a base, and supports the electron microscope sample support at an angle relative to an upper surface of the base. In any one of claims 5 to 7,
The transmission electron microscope is
A sample chamber;
a sample exchange chamber connected to the sample chamber via a gate valve;
a transport device that transports the cartridge between the sample exchange chamber and the sample chamber;
A sample observation method comprising: With the base,
a mounting portion provided on the base to which a sample support for an electron microscope is attached;
Including,
The base has a notch for irradiating a sample with a focused ion beam in a focused ion beam device, and a through hole for passing an electron beam in a transmission electron microscope.
was established,
The cartridge, wherein the cutout is provided with a beam portion spanning the cutout.

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