Sample carrier for use in charged particle microscope, and method of using such sample carrier in charged particle microscope

Abstract

To provide a method of preparing a sample in a charged particle microscope.SOLUTION: The disclosure relates to a method of preparing a sample in a charged particle microscope. The method comprises a step of providing a sample carrier having a mechanical support contour and a grid member connected thereto. The method comprises a step of connecting the sample carrier to a mechanical stage device of the charged particle microscope. Additionally, the method comprises a step of providing a sample such as a chunk-shaped or lamella-shaped sample and connecting the sample to the grid member of the sample carrier. The method allows, in an embodiment, easy and reliable transfer of a sample between a bulk sample and the sample carrier.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present embodiments relate to a sample carrier for use in a charged particle microscope and a method of using such a sample carrier in a charged particle microscope. The present embodiments further relate to a sample preparation device and method for an imaging system. More specifically, the present embodiments relate to a sample preparation stage with multiple degrees of freedom that enables in situ sample preparation and imaging. [Background technology]

[0002] Charged particle microscopy, particularly in the form of electron microscopy, is a well-known and increasingly important technique for imaging microscopic objects. Historically, the basic type of electron microscope has evolved into many well-known instrument types, such as transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning transmission electron microscopes (STEMs), as well as various subtypes, such as so-called "dual beam" instruments (e.g., FIB-SEMs) that additionally use a "machining" focused ion beam (FIB) to enable supporting activities such as ion beam milling or ion beam induced deposition (IBID).

[0003] Samples for electron microscopy imaging require specific preparation for observation under transmitted light or electron radiation. For example, thin slices (or sections) of the sample are typically cut or milled from the bulk sample in a grid or tube. Cutting or milling can be performed by a focused ion beam (FIB) system or within a dual-beam system that includes both an FIB and an electron microscope. Examples of such dual-beam systems include the Quanta 3D DualBeam system from FEI (Hillsboro, Oregon, USA). However, after preparing the thin slices using the FIB, the sample must be transferred to a platform suitable for imaging. Imaging with microscopes such as scanning transmission electron microscopes (STEM) may require positioning along multiple degrees of freedom to capture a suitable image.

[0004] Others have prepared STEM imaging stages with multiple degrees of freedom. For example, U.S. Pat. No. 7,474,419 describes a stage assembly for positioning a sample near a reference point. The stage assembly includes a sample table on which the sample can be mounted and a set of actuators arranged to effect translation of the sample table along directions substantially parallel to an X-axis perpendicular to a reference plane, a Y-axis parallel to the reference plane, and a Z-axis parallel to the reference plane. The X-, Y-, and Z-axes are mutually orthogonal and pass through the reference point. Furthermore, U.S. Pat. No. 6,963,068 describes a manipulator with a table that can move in five degrees of freedom: three perpendicular translations and two rotations.

[0005] However, techniques for manipulating samples for STEM or TEM analysis are more complex. These techniques require sample manipulation, both by FIB milling and carving, and subsequent STEM analysis to be performed at specific critical temperatures to prevent ice crystal formation within the sample or unwanted thawing of the sample between manipulations. Furthermore, positioning the lamella in the required orientation on the lamella support is extremely challenging. Thin lamellas, in particular, must be (nearly) coplanar with the lamella support to ensure proper placement in the (S)TEM for further examination. Furthermore, transferring the lamella from the sample to the support can involve welding and non-welding operations, which can damage or even destroy sample parts. For example, the lamella can fall off the needle or the support.

[0006] Therefore, what is needed is a system and method that allows for complex manipulation of a sample for STEM or TEM imaging without requiring so much sample processing that the sample is destructive.

[0007] US Patent Application Publication No. 2008 / 173813 A1 discloses a manipulator for rotating and moving a sample holder, where the sample holder having a grid on which the samples are placed is held by a manipulator that includes two members in the form of two tapered cylinders.

[0008] US Patent Application Publication No. 2020 / 273659 A1 describes a sample holder for a charged particle microscope, comprising a holder body with a recess for releasably receiving a sample carrier containing a sample. Summary of the Invention

[0009] According to one aspect, there is provided a method for preparing a sample in a charged particle microscope, as defined in claim 1. The method includes providing a sample carrier having a mechanical support contour and a grid member connected thereto, and connecting the sample carrier to a mechanical stage device of the charged particle microscope. As defined herein, the method further includes providing a sample, such as a sample in the form of a lamella or chunk, and connecting the sample to the grid member of the sample carrier. The step of connecting the sample to the grid member of the sample carrier is performed with the mechanical support contour and the grid member already connected to each other. In other words, the step of connecting the sample to the grid member of the sample carrier is performed after the step of providing a sample carrier having a mechanical support contour and a grid member connected thereto.

[0010] In contrast to prior art methods, the sample is connected to the grid element with the grid element and the mechanical support contour already connected. Instead of an indirect transfer, in which a lamella or chunk is placed on the grid element and the grid element is then connected to the mechanical support with the lamella, a direct transfer is possible. This saves time and reduces the risk of damaging the lamella or chunk, for example, when placing the grid element on the mechanical support. This is in contrast to prior art methods, in which the sample is first connected to the grid element and then the grid element / sample combination is connected to the mechanical support contour.

[0011] The method may include connecting the sample to a grid member of a sample carrier while the sample carrier is connected to the mechanical stage device.

[0012] As previously indicated, the grid member may include a half-moon grid, and / or the mechanical support contour may have a ring-shaped, annular, or substantially closed contour, as described above.

[0013] As previously indicated, the grid members may be integrally connected to the mechanical support contours.

[0014] In one embodiment, the method may be performed in a charged particle microscope, including a focused ion beam microscope.

[0015] In one embodiment, providing the sample comprises providing a bulk sample and using the focused ion beam to create the sample, such as a lamellar-shaped sample or a chunk-shaped sample, in the bulk sample.

[0016] The method may include the steps of releasing the sample from the bulk sample and transferring the sample to the sample carrier.

[0017] An elongated transport member may be used to transport the sample.

[0018] In one embodiment, the elongate transport member defines an angle with a plane defined by the sample carrier, and the angle is maintained substantially constant during at least a substantial portion of the transfer of the sample to the sample carrier. The angle is particularly maintained constant during the transfer from the bulk sample to the sample carrier. Note that releasing the sample from the bulk sample and / or attaching the sample to the sample carrier may involve a change in the angle. A substantial portion is considered to be at least 80%, preferably at least 90%, or more preferably at least 95% of the distance traveled between the bulk sample holder and the grid sample holder.

[0019] In one embodiment, translational motion is substantially used to transport said sample.

[0020] The translational movement may be provided by a relative movement between an elongated manipulator and a mechanical stage of the charged particle microscope. In particular, the elongated manipulator may move relative to the mechanical stage. It is conceivable that during the transfer, some mechanical stage movements may also be performed.

[0021] In one embodiment, the elongate transport member comprises a needle, which provides for easy and rapid delivery, although it is envisioned that other shapes may also be used.

[0022] The methods defined herein may be carried out in a cryogenic environment.

[0023] The method may be performed on a multi-axis sample preparation stage including a bulk sample holder configured to rotate a sample position about a first bulk axis parallel to the orientation of the bulk sample holder and to rotate the sample position about a bulk flip axis perpendicular to the orientation of the bulk sample holder, and a grid sample holder for holding a sample grid adjacent to the bulk sample holder, the grid sample holder configured to rotate the sample grid about a first grid axis parallel to the orientation of the grid sample holder and a grid flip axis perpendicular to the orientation of the grid sample holder.

[0024] This method can also be performed with a sample preparation stage including a bulk sample holder and a grid sample holder, where the sample preparation stage is rotatable around a bulk flip axis perpendicular to the orientation of the bulk sample holder. Both the bulk sample holder and the grid sample holder can be assumed to be fixed relative to one another, i.e., rotating the preparation stage causes both the bulk sample holder and the grid sample holder to move in the same manner. The rotation axis (bulk flip axis) can be in the XZ plane, and the elongated manipulator can also extend substantially within the XZ plane. Note that the bulk sample holder and the grid sample holder can be angled relative to the base plate of the preparation stage, i.e., positioned at an angle relative to the XY plane. This angle can be, for example, approximately 45 degrees.

[0025] The method can also be performed in a dual beam system having a focused ion beam and a scanning electron microscope, which can include a multi-axis sample preparation stage as described above.

[0026] The methods defined herein may be performed in situ by providing a multi-axis sample preparation stage as described above, preparing the sample by cutting a lamella or chunk from a bulk sample stored in a bulk sample holder, and transferring the lamella from the bulk sample holder to a grid member of a sample carrier provided in a grid sample holder.

[0027] According to one aspect, the present invention provides a sample carrier for use in a method as defined herein and as set forth in claim 13. The sample carrier comprises a mechanical support contour arranged to be connectable to a mechanical stage device of a charged particle microscope. The sample carrier further comprises a grid member connected to the mechanical support contour and arranged to hold a sample, in particular a sample emanating from a bulk sample. The sample may be, for example, a so-called chunk or lamella, although other shapes and forms are also conceivable. More generally, embodiments include any relatively small sample pieces collected from a relatively large bulk sample. As defined herein, the grid member is arranged to receive the sample in an assembled state of the sample carrier.

[0028] The grid member may be, for example, a so-called half-moon grid, also called a half-moon grid lift-out TEM sample holder for FIB applications (FIB lift-out grid), as described, for example, in U.S. Pat. No. 9,159,531 B2.

[0029] The mechanical support contour can have, for example, a ring-shaped, annular, or substantially closed contour. The mechanical support can be, for example, a so-called Cryogenic FIB AutoGrid available from Thermo Fisher Scientific, FEI, Hillsboro, Oregon, USA.

[0030] As defined herein, the grid element is pre-connected to the mechanical support contour, and the grid element does not contain any sample in the pre-connected state. One advantage of the sample carrier according to the invention is that a lamella or chunk can be placed on the grid element (also called a lamella support) while the grid element is already placed inside the mechanical support or at least pre-connected to it. Instead of an indirect transfer, in which the lamella or chunk is placed on the grid element and the grid element is connected to the mechanical support by the lamella, a direct transfer is possible. This saves time and reduces the risk of damaging the lamella or chunk, for example, when placing the grid element on the mechanical support.

[0031] As previously mentioned, the present invention is useful for transferring lamellae or chunks originating from a larger bulk sample. However, the present invention is not limited to any particular shape or form of the lamellae or chunks that are transferred to the pre-connected sample carrier.

[0032] A sample carrier may comprise the lamellar-shaped or chunk-shaped sample, which may only be connected to the sample carrier when the sample carrier, including the grid member pre-connected to the mechanical support contour, is mounted on the mechanical stage device of the charged particle microscope.

[0033] In one embodiment, the grid member and the mechanical support contour are integrally formed. This means that the grid member and the mechanical support can be integrally connected, i.e., fixedly connected to each other. For example, the grid member and the mechanical support can be fixedly connected to each other during the manufacturing process, which can include, for example, molding techniques or techniques based on MEMS manufacturing. In this way, the grid member and the mechanical support contour are already fixedly connected to each other and no prior clipping procedure needs to be performed. An integrally formed sample carrier can be advantageously used in the methods disclosed herein, which are described next. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 is a perspective view of one embodiment of a base stage having a bulk stage and a grid stage. [Figure 2] FIG. 1B is an enlarged view of the bulk stage and grid stage of FIG. 1A. [Figure 3] FIG. 4 is an enlarged view of the prepared sample of FIG. 3. [Figure 4] FIG. 1 is a flow diagram of one embodiment of a method for in situ preparation of a sample for imaging. [Figure 5] FIG. 1 is a close-up view of the setup for preparing samples as disclosed herein. [Figure 6] FIG. 1 is a perspective view of one embodiment of a heating / cooling element via a bearing. [Figure 7a] 1A-1C are different views of the stage connected to the thermal control system. [Figure 7b] 1A-1C are different views of the stage connected to the thermal control system. [Figure 8] FIG. 1 is a diagram of a simple yet effective structure for bulk sample preparation and subsequent transfer of the bulk sample to a sample carrier as defined herein. DETAILED DESCRIPTION OF THE INVENTION

[0035] Embodiments of the present invention relate to sample carriers, sample processing systems, and methods for preparing samples for imaging in charged particle microscopes, such as electron microscopes. The sample carrier may be used in a sample preparation and imaging stage of a dual-beam electron microscope with multiple sample positions and the ability to tilt each sample position about multiple axes. One embodiment of a multi-axis stage includes a bulk preparation stage for manipulating bulk samples, for example, by performing focused ion beam processing of the bulk sample to mill or slice lamella-shaped samples of the bulk sample for further imaging. The multi-axis stage may include holders for grids, tubes, planchets, or TEM lift-out grids to process various sample types. In particular, the holder may be part of a mechanical stage device of the charged particle microscope that is configured to connect to the sample carrier, as defined herein. Furthermore, the multi-axis stage may be configured to move in multiple directions with multiple degrees of freedom to position the bulk sample under the FIB for appropriate milling or slicing of the bulk sample.

[0036] In addition to the bulk preparation stage, embodiments of the multi-axis stage also include a grid stage configured to hold thin structures (e.g., lamellae) of the bulk sample mounted on a grid for imaging. Thus, once cut, lamellae from the bulk sample can be transferred from the bulk stage to the grid stage, for example, by using elongated transport members in the form of manipulator needles. In some embodiments, the grid stage can move in multiple dimensions, enabling dual-axis tomography with multiple viewing angles. Because the bulk stage and grid stage are on the same multi-axis stage, a single multi-axis stage can be used to cut the lamella and perform TEM scans on the cut lamella sections without venting the chamber used to transfer the lamella to the grid stage. Thus, the multi-axis stage can include a bulk stage with multiple degrees of freedom and a grid stage with multiple degrees of freedom along various axes that allow the components of the system to move in multiple dimensions relative to each other.

[0037] The multi-axis stage can be configured to perform embodiments of the methods defined herein, including providing a sample carrier as defined herein, the sample carrier having a mechanical support contour and a grid member connected thereto, and in particular connecting the sample carrier to a mechanical stage device of the charged particle microscope by connecting the sample carrier to a grid stage of the multi-axis stage. This also includes providing a sample, such as a lamellar-shaped sample or a chunk-shaped sample taken from a bulk sample provided on a bulk stage, and connecting the sample to the grid member of the sample carrier. In particular, when an elongated transport member such as a manipulator needle is used to transfer the lamellar-shaped sample, the multi-axis stage also enables the release of the lamellar-shaped sample or chunk-shaped sample from the bulk sample and the transfer of the lamellar-shaped sample to the sample carrier. The multi-axis stage further allows for easy transfer of lamellar shaped samples to the sample carrier, for example because during transfer of the lamellar shaped sample to the sample carrier, the elongated transport member defines an angle with a plane defined by the sample carrier, and the angle is kept substantially constant by a translational movement. Thus, the mechanical stage of the charged particle microscope, in particular the multi-axis stage, allows for providing a translational movement.

[0038] It should be understood that many different types of processing can be applied to a sample, as described below. Embodiments of the present invention include any type of processing that can be used to prepare a sample for transmission electron microscopy (TEM) or scanning transmission electron microscopy (STEM) analysis. For example, lamellae or chunks can be created from sites in a bulk sample. In this system, a bulk stage holds the bulk sample in which the lamellae or chunk sites are located. The lamellae or chunks are then prepared in the bulk stage.

[0039] Furthermore, it should be understood that the present embodiments are not limited to any particular configuration of microscope. For example, any type of microscope used to capture images of a sample is within the scope of the present embodiments. Such microscopes include, for example, visible light microscopes, confocal microscopes, and infrared and near-infrared microscopes. Those skilled in the art will recognize that the embodiments illustrated herein with respect to electron microscopes can be readily adapted to other types of microscopes.

[0040] In another embodiment, a lift-out procedure can be performed. In this system, a manipulator can transfer a lamella or chunk located on a bulk stage to a grid of sample carriers provided on a grid stage. In some embodiments, the lamella can be further processed after the manipulator is removed. For example, the lamella can be milled from a thick lamella to a thin lamella using a focused ion beam.

[0041] Lamella preparation includes processes such as identifying lamella sites (including multiple cross sections of the sample), protective deposition (e.g., coating the lamella sites with a metal cap layer), adding fiducial markers, rough milling (e.g., to create a lamella of approximately 2 µm), medium milling (e.g., to thin the lamella to approximately 250-400 nm), fine milling (e.g., to thin the lamella to its final thickness), undercutting to release the sample from the substrate, endpoint, lamella cleaning (e.g., low kV cleaning), and / or sample transfer.

[0042] The systems and methods disclosed herein can be used for sample preparation of cryogenically frozen samples. For example, these methods can be performed without venting the microscope chamber. FIB preparation can be used with cryogenically frozen samples in appropriately equipped instruments, such as dual-beam microscopes, allowing for cross-sectional analysis of liquid- or fat-containing samples, such as biological samples, pharmaceuticals, foams, inks, and foods. As described in more detail below, the system can further include temperature control elements for maintaining the temperature within the system. Thus, in some embodiments, the methods disclosed herein can be performed at room temperature, elevated temperatures, and / or cryogenic temperatures.

[0043] As used herein, the term "sample" can include any type of sample from an organism, but typically includes tissue, cells, viruses, cell structures, or any other biological sample of interest.

[0044] Samples can be prepared for electron microscopy, which is used in materials science applications such as semiconductor materials and polymers. Electron microscopy can also be used in biology and life sciences for applications such as diagnostic electron microscopy, cryobiology, protein localization, electron tomography, cellular tomography, cryo-EM, toxicology, biological production and viral load monitoring, particle analysis, pharmaceutical quality control, structural biology, 3D tissue imaging, virology, and vitrification. These separate types of applications can be performed by a variety of different types of electron microscopes, including, for example, transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), reflection electron microscopes, scanning and transmission electron microscopes, and low-voltage electron microscopes. Sample Preparation System Overview

[0045] 1 is a perspective view of one embodiment of a multi-axis stage 100. The multi-axis stage 100 includes a circular base 101 that supports a rectangular stand 103. Attached to the left end of the rectangular stand 103 is a vertical wall 104 that holds a bulk stage 110. The bulk stage 110 is movably mounted to the vertical wall 104 and is used to hold a bulk sample.

[0046] Bulk stage 110 includes a bulk rotation actuator 115 configured to rotate sample holder 118 circumferentially about bulk rotation axis 112, as shown. Movement of actuator 115 thus results in rotational movement of the bulk sample within sample holder 118, with 360 degree movement about the Y axis of multi-axis stage 100. This allows the sample to rotate along an axis parallel to the orientation of sample holder 118.

[0047] In addition to this rotational motion, bulk stage 110 also has multiple degrees of freedom about bulk flip axis 111, which uses flip actuator 119 attached to vertical wall 104 to rotate sample holder 118 about the X-axis to "flip" the sample from the front of base stage 100 to the rear of multi-axis stage 100, and vice versa. This allows bulk sample holder 118 to rotate about a line perpendicular to the orientation of the sample holder.

[0048] Rotation of the bulk sample around the bulk rotation axis 112 enables sample processing, such as focused ion beam processing, resulting in a rotationally symmetric sample around the rotation axis. This can eliminate or reduce anisotropy / inhomogeneity in the sample after ion beam processing. This angular degree of freedom (DOF) around the rotation axis, along with the additional angular DOF provided around the bulk flip axis 111, allows a wide range of specific crystallographic orientations within the sample to be oriented along the first and / or second illumination beams. Thus, combining the angular DOF around the rotation and flip axes can enable both α-tilt and β-tilt of the sample.

[0049] In certain embodiments, the angular stroke of the sample holder 118 around the bulk flip axis is substantially 360 degrees or more. When the flip axis is arranged parallel to the main axis of the multi-axis stage 100 (by appropriate angular adjustment of the stage assembly around the rotation axis) and the focused beam is an ion beam, such angular stroke allows the stage assembly to be used as a kind of "ion lathe." Such a setup can produce various precision items, such as tips or probes, that require a specific cylindrical / conical profile around the flip axis. In a similar way, a "laser lathe" can be realized using a laser beam as a second illumination beam.

[0050] Mounted on rectangular stand 103 and directly adjacent to bulk stage 110 is grid stage 150. Like the bulk stage, because grid stage 150 is mounted on rectangular stand 103, it also provides the sample with multiple degrees of freedom around several axes. As shown in FIG. 1, grid stage 150 has a grid flip axis 151 and a grid rotation axis 152, which allow grid holder 156 to move in multiple X and Y dimensions. Rotational motion along grid rotation axis 152 is controlled by grid rotation actuator 154, which moves grid holder 156 around the Y axis of multi-axis stage 100. This allows the grid holder to rotate in a direction parallel to grid holder 156.

[0051] Rotational motion along grid flip axis 151 is controlled by flip actuator 155, which allows the grid holder to rotate about the X-axis of multi-axis stage 100. This allows the grid stage to rotate in a direction about the Y-axis of multi-axis stage 100. This multi-axis rotational motion provides several degrees of freedom for a sample placed on grid holder 156 to provide for imaging of the sample.

[0052] By mounting the bulk stage and grid stage adjacent to each other on the same base, this single stage can be used to prepare samples using an ion beam and then image those samples with an electron beam. In particular, the single stage can be used with lamellar-shaped samples. In dual-beam devices, the multi-axis stage can be placed within the device and then used for sample preparation and imaging without removing the multi-axis stage from the dual-beam device. Furthermore, as described below, the sample can be kept at a desired temperature by cooling or heating the multi-axis stage to the desired temperature and performing all sample manipulations within the dual-beam system without having to expose the stage and sample to room temperature conditions.

[0053] At the rear of the multi-axis stage 100 is a STEM stage 160 that allows lateral movement 161 of a detector holder 162 for performing scanning transmission electron microscopy on a sample using the same multi-axis stage 100. In some embodiments, the aforementioned components can move about axes independently of each other.

[0054] At the rear of the multi-axis stage 100, a retractable S / TEM detector may be placed in a detector holder 162 that may be retracted by a retractor 160. The detector may also be protected with a cover to avoid, for example, radiation or chemical damage. In some embodiments, a cover may be used as an alternative to retractability of the S / TEM detector.

[0055] 2 is an expanded view of one embodiment of the bulk stage 110 and grid stage 150, showing their relationship to one another. The bulk stage includes a bulk arm 210 configured to hold a bulk sample carrier 215 with a bulk sample 220. The bulk arm 210 can rotate about the bulk stage such that the orientation of the bulk sample 220 changes. For example, the bulk arm 210 can rotate about a bulk rotation axis 112 to rotate the bulk sample 220. The bulk arm 210 can also flip about a bulk flip axis 111 to flip the bulk sample 220.

[0056] Adjacent to the bulk stage 110 is the grid stage 150, which is shown with a grid arm 250 configured to hold a sample carrier 255 configured to hold a sample. The grid arm 250 can move around the grid stage with multiple degrees of freedom so that the orientation of the sample carrier 255 can change over time and during electron microscopy. For example, the grid arm 250 can rotate about a grid rotation axis 152. The grid arm 250 can also flip about a grid flip axis 152 to flip the grid plate.

[0057] As shown in Figure 2, a manipulator 270 and gas supply system 280 can be used to transport a lamella harvested from a sample 220 in the bulk stage 110 and move the lamella to the grid stage 150 by methods known in the art. As shown in Figure 3, the lamella 410 can be attached to a sample carrier 255 including a mechanical support contour 310 and a grid member 320 for further analysis.

[0058] As proposed herein, lamellar sample transfer to the sample carrier 255 may occur by providing a sample carrier 255 having the mechanical support contour 310 and the grid member 320 connected thereto, and placing the sample carrier 255 on the grid stage. The method proposed herein, in one embodiment, further comprises providing a bulk sample and creating a lamellar-shaped sample in the bulk sample, for example by the FIB.

[0059] Next, as shown in FIG. 4, the bulk stage can be positioned so that the bulk sample is disposed parallel to the XY plane of the stage (see FIG. 1 for XYZ coordinates). The sample carrier can be positioned so that the substantially planar grid member is disposed substantially parallel to the XY plane of the stage. The stage can then be tilted, for example, by about 10 degrees about the x-axis and rotated, for example, by about 10 degrees about the z-axis. This allows the lamellar-shaped sample to be transferred from the bulk sample to the sample carrier by an elongated transport member, such as a transfer needle, through translational movement of the stage (i.e., movement in the XY plane) without a mechanical support profile blocking the elongated transport member. The methods described herein allow the elongated transport member to define an angle with a plane defined by the sample carrier and maintain the angle substantially constant during the transfer of the lamellar-shaped sample to the sample carrier. The lamellar-shaped sample can then be transferred using pure translational movement, for example, provided by a multi-axis stage.

[0060] In another embodiment, shown in FIG. 8 , the stage 600 simply includes a rotation axis that lies in the ZX plane, allowing the elongated manipulator 270 to be positioned at an angle relative to the plane defined by the bulk sample carrier 110 and / or the sample carrier 150. This allows the elongated manipulator 270 to pick up a lamella or chunk from the bulk sample and move it toward the sample carrier 150, such that the elongated manipulator avoids contact with the mechanical support contours of the sample carrier. In the embodiment shown in FIG. 8 , the bulk sample holder 110 and the grid holder 150 are rigidly connected to each other, meaning that relative movement between the two is prevented. The grid holder 150 and the bulk sample holder 110 are provided at 45 degrees relative to the XY plane. The bulk sample holder 110 and the grid holder 150 are substantially in the same plane. The embodiment of FIG. 8 is a simple yet effective structure for bulk sample preparation and subsequent transfer of the bulk sample to a sample carrier as defined herein.

[0061] Of course, it should be understood that the present embodiment is not limited to a particular configuration of bulk sample carrier 215, so long as it includes a grid member and a mechanical support contour. The mechanical support contour substantially surrounds the circumference of a grid member, particularly a substantially planar grid member, such that the mechanical support contour at least partially blocks line of sight to the grid member. In principle, any type of sample carrier used to hold samples and enable preparation as disclosed herein is within the scope of the present embodiment. Similarly, it should be understood that the present embodiment is not limited to a particular configuration of grid plate 310. For example, any type of plate used to hold samples and enable further processing and / or imaging as disclosed herein is within the scope of the present embodiment. While the embodiment includes the aforementioned half-moon plate, other plates are similarly envisioned. Exemplary Methods of Sample Preparation

[0062] 4 shows a flowchart illustrating an exemplary process 500 that may be performed within one implementation of the sample preparation system 100. Process 500 begins at block 502, where a bulk sample is loaded onto the bulk stage. After the bulk sample is loaded onto the bulk stage, the process moves to block 504, where a lift-out grid is loaded onto the grid stage. Next, process 500 moves to block 506, where the bulk sample is centered on the bulk stage. For example, the bulk sample can be positioned by rotating the bulk sample arm about a bulk rotation axis and / or flipping the bulk sample arm about a bulk flip axis to properly position the bulk sample to create the desired lamella.

[0063] Once the bulk sample is positioned, process 500 moves to block 508, where a protective metal layer is locally deposited on the bulk sample. It should be understood that in other embodiments, the order of blocks 508 and 510 can be reversed. As described herein, the protective metal layer can comprise any material, such as platinum or tungsten, although other materials are also contemplated. It should be noted that sputter coating may be used prior to block 508, as is known in the art. Next, process 500 moves to block 510, where a region of interest on the bulk sample is determined by the user. The region of interest may comprise, for example, one or more discrete features and / or one or more reference features, such as fiducial markers in the deposited protective metal layer. Alternatively, the region of interest can be marked by previously placing the sample under an optical microscope and using a laser marker to indicate the region of interest.

[0064] Once the region of interest is identified, process 500 moves to block 512, where a lamella is cut. As described herein, the lamella may be positioned to optimize the characteristics of the location of interest. In some embodiments, a thick lamella is cut. In one embodiment, the lamella is cut using a focused ion beam aimed at cutting the desired lamella from the bulk sample. As can be appreciated, the desired region is properly cut by using the multiple degrees of freedom provided by the bulk stage, as described above. Once the lamella is cut, the operator transfers the lamella from the bulk stage to the grid stage.

[0065] For this purpose, the setup shown in FIG. 5 can be used. Here, it can be seen that the bulk sample 215 is provided on the bulk stage 210 and the sample carrier 255 is connected to the sample stage 150. A needle-shaped elongated manipulator 270 is shown and can be used to transfer the lamellar-shaped sample from the bulk sample to the sample carrier. As shown in FIG. 5, the sample carrier 255 has a mechanical support contour 310 by which the sample carrier 255 is connected to the grid stage 150. The sample carrier 255 also has a grid member 320, such as a half-moon grid, connected to the mechanical support contour 310 and arranged to receive the lamellar-shaped sample. In the embodiment shown in FIG. 5, the sample carrier 255 is connected to the grid stage 150 so that the plane defined by the sample carrier 255 is arranged substantially perpendicular to the plane defined by the bulk sample 215 connected to the bulk stage 210. In other words, if the bulk sample 215 is primarily positioned in the XY plane, the sample carrier 255 is primarily positioned perpendicular to the XY plane, e.g., parallel to the ZX plane. As shown in FIG. 5, the elongated manipulator is positioned at an angle relative to the plane defined by the sample carrier so that it can approach the grid member 330 without colliding with the mechanical support contour 310 of the sample carrier 255. This setup allows for quick and easy transfer of lamellar-shaped samples from the bulk sample to the sample carrier, and can be performed using purely translational motion of the multi-axis stage 100 relative to the manipulator needle. This will be explained in more detail by returning to block 514 of FIG. 4.

[0066] As an example, in block 514, an elongated manipulator is inserted into the bulk sample, and in block 516, the manipulator is attached to a lamella cut from the bulk sample. The manipulator may be, for example, a needle or other device configured to attach to the lamella. For example, the manipulator may be temporarily attached to the lamella to enable transfer between the bulk stage and an adjacent grid stage. The manipulator then extracts the lamella in block 518. The manipulator is then retracted from the bulk sample in block 520.

[0067] Next, process 500 moves to block 522, where the grid stage with the sample carrier can be centered by translational movement of the multi-axis stage. The manipulator can then approach the sample carrier and attach a lamella to the grid member of the sample carrier. Process 500 moves to block 528, where the manipulator is removed from the lamella. For example, the FIB can be used to cut the manipulator needle from the lamella. The manipulator can then be stored at block 530.

[0068] As described herein, in some embodiments, the lamella can be thinned from a thick lamella to a thin lamella, for example, process 500 moves to block 532, where a focused ion beam thins the thick lamella to a thin lamella.

[0069] After the lamella is transferred to a sample carrier on a grid stage and thinned to the desired thickness, process 500 moves to decision block 540 to determine whether the lamella will be inspected in a compact dual beam device (SDB). If a decision is made not to remain in the SDB device, multi-axis stage 100 is unloaded in block 560 and may be transferred, for example, to a TEM system in block 565. However, if a decision is made to remain in the SDB device, the sample may be directly STEM imaged. For example, the sample may undergo STEM tomography in block 550 before being unloaded in block 555. In another example, the sample may undergo STEM imaging in block 545 before being unloaded in block 555.

[0070] It should be noted that embodiments of the present invention also cover variations of the method, such as, for example, steps 502 and 504 being swapped, or blocks 510 and 508, as would be apparent to one skilled in the art. Multi-axis stage temperature control

[0071] In some embodiments, the system can further include a thermal control system configured to control the temperature of the multi-axis stage 100. As shown in FIG. 6, a thermal control system 600 can be used to heat or cool the multi-axis stage 100 while still allowing the stage 100 to move circularly within the imaging system. The thermal control system 600 includes a base 601 that is attached via a platform 602 through a series of connectors 604. A system of standoffs 603 can be used to raise the platform 602 to a desired level within the imaging system. The standoffs are attached to the platform 602 via a series of pins 608.

[0072] As shown, the base 601 fits within a cylindrical sleeve 610, which fits into the center of a metal ring 612. Fitting onto the top surface 614 of the sleeve 610 is a heat transfer body 620 including heat transfer pipes 616 configured to move a heat transfer medium. In one embodiment, the heat transfer medium is cooled with dry or liquid nitrogen, and the temperature of the heat transfer plate 620 can be controlled by controlling the flow rate of the dry nitrogen with a flow meter (not shown) or by adding a supplemental heat source, such as a thermal resistor. Thus, by controlling the type and amount of heat transfer medium circulating through the heat transfer pipes 616 (and / or by controlling the additional heat source), a user can control the resulting temperature of the heat transfer plate 620.

[0073] Residing on top of and in thermal contact with heat transfer plate 620 is a bearing ring 630 having a plurality of slots 635. Each of the slots 635 in bearing ring 630 is configured to hold a thermally conductive roller 640. On top of rollers 640 is top plate 650. Top plate 650 includes a mounting bracket 660 and a centering pin 665 designed to mount with multi-axis stage 100 and provide thermal heating or cooling functionality to multi-axis stage 100. Top plate 650 can rotate about its axis driven via base 601.

[0074] As can be imagined, when the multi-axis stage is attached to the mounting bracket 660, the stage can rotate 360 ​​degrees on roller bearings (e.g., ball bearings or needle bearings) and maintain thermal connection with the heat transfer medium flowing through the pipes 616. In this embodiment, all components can be designed with high thermal conductivity. For example, the roller bearings can be made of steel with a conductivity of 46 W / mK. The cooling stage components can be made of oxygen-free copper or other materials with high thermal conductivity, such as gold. In some embodiments, the temperature of the shuttle receiver can drop to -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, or -180°C or lower. In some embodiments, a device can be used to transfer heat so that the system is heated rather than cooled by pumping a heated liquid or gas through the pipes 616.

[0075] It should be noted that the stage may be equipped with one roller bearing that provides the necessary mechanical support and degrees of freedom, while the stage further exhibits a second roller bearing that thermally connects the stage to a stationary cooling body, for example cooled by liquid nitrogen.

[0076] 7a and 7b show different views of stage 100 connected to thermal control system 600 as described above. Stage 100 may be a multi-axis stage, as described with reference to FIG. 1. In the embodiment shown, stage 100 includes bulk stage 110 and grid stage 150. Stage 100 is connected to thermal control system 600 in that stage 100 is provided on top plate 650 and connected to mounting bracket 660 and centering pin 665 (see FIG. 7b).

[0077] As shown, needles 270 can be used to place lamellar-shaped samples on sample carriers 255, which have a grid member connected, e.g., integrally connected, to a mechanical support contour. The sample carrier defines a plane and is positioned substantially perpendicular to the bulk sample carrier. Bulk sample carrier 215 is positioned substantially parallel to the surface of top plate 650. In the embodiment shown in Figures 7a and 7b, needles 270 are positioned at an angle relative to bulk sample carrier 215 (i.e., an angle relative to the xy plane) and at an angle relative to sample carrier 255 (i.e., an angle relative to the zx plane). This allows for quick and easy transfer of lamellar-shaped samples from bulk sample carrier 215 to sample carrier 255, for example, by simple translational movement of stage 100. Detecting the frozen state of ice

[0078] Another embodiment relates to the determination of the vitreous ice state in a sample. This typically relies on TEM electron diffraction images, typically acquired in a region of interest on a thin section of a frozen sample. The vitreous ice state helps preserve the natural structural morphology of biological cell membranes and dispersed particles. In contrast, crystalline ice destroys their structure and distribution. While ring patterns indicate that the ice is amorphous (vitreous), spot patterns indicate the presence of hexagonal or cubic crystalline structures. Very often, the vitreous state results in diffraction rings that are too sharp or too blunt, or it can be unclear whether they were produced by a W, LaB6, or FEG TEM electron gun.

[0079] When preparing a TEM section, one would like to know whether the surrounding ice is crystalline or glassy at that point, which helps the user decide whether it is useful to continue with that sample or start with a new one.

[0080] An alternative embodiment is a method that can be used with a field emission gun SEM or SEM for samples where it is important to keep the temperature below the glass transition temperature of 136 K (-137 °C). In this embodiment, an ice sample is FIB milled to a desired thickness so that it can be viewed by an analytical detector from below using an electron beam at a desired voltage when rotated and tilted relative to the horizontal. By tilting the ice sample at a positive or negative angle from the horizontal, the analytical detector can observe different transmitted orientation contrasts. This different orientation contrast, if present, comes from the lattices of differently oriented crystals within the thin sample. If no crystalline form is present because the sample is glassy, ​​the contrast remains constant as the sample is tilted.

[0081] This allows detection of whether a sample has formed crystalline ice. This is because the glassy state is a state of random atomic structure and does not display orientation contrast. This is a very direct and reliable method of determining the ice state of a sample if it is prepared immediately in a FEG SEM without further transfer to a cryo-TEM system, where hexagonal ice contamination could compromise the results. With improvements in SEM and dual-beam instruments to resolve sub-nanometer resolution, there is often little need to transfer to a TEM for direct-resolution imaging or angular tomography. Therefore, this method is useful for confirming the state of segmented ice within an SEM or dual-beam instrument. equivalent

[0082] The foregoing specification is considered sufficient to enable one skilled in the art to practice the present embodiments. The foregoing description and examples detail certain preferred embodiments and explain the best mode contemplated by the inventors. However, no matter how detailed the above appears, it will be understood that the present embodiments can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0083] The methods and sample carriers are often described using lamellar-shaped samples. However, in principle, the embodiments described herein are also applicable to chunk-shaped samples, or samples of other shapes. More generally, the invention described herein is applicable to any sample originating from a larger bulk sample.

[0084] The grid member, in one embodiment, comprises a half-moon shaped grid, although it should be noted that other grid shapes and grid types are possible and the invention is not limited to the use of half-moon shaped grids. Other shapes may also include ring-shaped, annular, or substantially closed contour shaped grids.

[0085] The term "comprising" as used herein is intended to be open-ended, encompassing not only the recited elements, but any additional elements as well.

[0086] The desired protection is determined by the appended claims.

Claims

[Claim 1] 1. A method of preparing a sample in a charged particle microscope, the method comprising: - providing a sample carrier having a mechanical support contour and a grid member connected thereto; - connecting said sample carrier to a mechanical stage device of said charged particle microscope; providing a sample, wherein the sample is processed by focused ion beam processing and is moved by a manipulator from the bulk stage to the grid stage and fixed to the grid stage, with the bulk stage and the grid stage mounted on the same base; - connecting the sample to the grid member of the sample carrier, with the mechanical support contour and the grid member pre-connected to each other, the sample carrier is connected to the grid stage such that a plane defined by the sample carrier is disposed perpendicular to a plane defined by a bulk sample connected to the bulk stage; How to prepare samples for charged particle microscopy.

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