Lamella preparation from thick hpf samples

Abstract

To provide a preparation technique of an observation sample for a transmission microscope applicable to a cryogenic sample having a thickness exceeding 30 micrometers.SOLUTION: An ion beam is directed toward a first surface of a sample for a first exposure of the first surface. The sample defines a first surface and a second surface opposite the first surface. The sample is rotated by an angle β with respect to the beam axis B of the charged particle beam system to orient the second surface to receive the ion beam. The ion beam is directed toward a second surface of the sample. The sample is rotated relative to the beam axis B to orient the first surface to receive the ion beam. The ion beam is directed toward the first surface of the sample for a second exposure of the first surface.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of provisional patent application serial number US63 / 664 / 390, filed June 26, 2024, entitled "LAMELLA PREPARATION FROM THICK HPF SAMPLES," the contents of which are incorporated herein by reference in their entirety.

[0002] [Technical field] FIELD OF THE DISCLOSURE Embodiments of the present disclosure relate to charged particle microscope systems, and algorithms and methods for their operation. In particular, some embodiments relate to techniques for preparing samples for examination in a charged particle beam microscope. [Background technology]

[0003] Cryo-FIB / SEM combined with cryo-electron tomography (cryo-ET) has emerged within the field of cryo-electron microscopy (cryo-EM) as a method for obtaining in situ the highest resolution structural information of complex biological samples in natural and non-native environments. However, challenges remain with traditional cryo-FIB / SEM workflows, including milling thick samples with vitrification issues, samples with preferred orientation, low throughput when milling small and / or low-concentration samples, and samples with poor distribution on the grid squares.

[0004] To address these challenges, a general approach called the "waffle method" has been developed that utilizes high-pressure freezing techniques. While the waffle method alleviates some of these challenges, as well as geometric and other limitations, it is limited to samples with thicknesses of 30 micrometers or less. Therefore, a different sample preparation technique that addresses the same challenges and is applicable to cryogenic samples with thicknesses greater than about 30 micrometers is needed. Summary of the Invention

[0005] The terms and expressions used are used as terms of description without limitation, and the use of such terms and expressions is not intended to exclude equivalents of the illustrated and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed subject matter. Thus, while the claimed subject matter has been specifically disclosed by embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present disclosure as defined by the appended claims. For example, the above-described aspects and various embodiments can be combined with one or more other aspects and / or embodiments of the same or other aspects.

[0006] In a first aspect, a method of preparing a sample for inspection by a charged particle beam system includes directing an ion beam toward a first surface of the sample for a first exposure of the first surface. The sample can define a first surface and a second surface opposite the first surface. The method can include rotating the sample by an angle β relative to a beam axis B of the charged particle beam system to orient the second surface to receive the ion beam. The method can include directing the ion beam toward the second surface of the sample. The method can include rotating the sample relative to the beam axis B to orient the first surface to receive the ion beam. The method can also include directing the ion beam toward the first surface of the sample for a second exposure of the first surface. Cut.

[0007] In some embodiments, the method further includes forming a trench in the first surface using the ion beam. The trench can be a first trench. The method can further include forming a second trench in the second surface. The second trench can be substantially aligned with the first trench. The first trench and / or the second trench can be angled trenches oriented at a non-zero angle relative to the first surface. The angled trench can be oriented at a non-zero angle relative to a normal vector of the first surface. The first trench can extend partway through the sample. The second trench can extend through the sample.

[0008] In some embodiments, the magnitude of the angle β is about 180 degrees. The method can include rotating the sample by an angle α relative to the beam axis B and orienting the first surface to receive the ion beam. The magnitude of the angle α can be about 10 degrees to about 60 degrees. Directing the ion beam toward the second surface can include orienting the second surface to receive the ion beam at an angle γ relative to the second surface. Directing the ion beam toward the second surface can define a facet oriented substantially at the angle γ relative to the first surface.

[0009] In some embodiments, the method further includes depositing a protective layer on at least a portion of the facet. The protective layer may include a metal and / or a dielectric. Depositing the protective layer may include decomposing a precursor using an ion beam. The sample may include a region of interest. The method may further include forming a fin from the facet using the ion beam and thinning the fin to form a lamella including at least a portion of the region of interest. The region of interest may be a first region of interest. The sample may include a second region of interest. The method may further include forming a first lamella from the fin including at least a portion of the first region of interest and forming a second lamella from the fin including at least a portion of the second region of interest.

[0010] In some embodiments, the method further includes forming a stress relief cut in the facet between the first region of interest and the second region of interest.

[0011] The sample may be a cryogenically frozen sample. The sample may include biological material prepared by high pressure freezing (HPF).

[0012] The method can further include milling a first vertical trench through the sample, extending from the first surface to the second surface, the first vertical trench being substantially perpendicular to the first surface. The method can further include milling a second vertical trench through the sample, extending from the first surface to the second surface, the second vertical trench being substantially perpendicular to the first surface and / or substantially aligned with the first vertical trench. The first and second vertical trenches can be formed on either side of a region of interest in the sample from which the lamella is to be prepared.

[0013] In a second aspect, a charged particle beam system includes a charged particle source. The system can include a sample stage operatively coupled to a column of the charged particle beam and configured to translate and / or rotate a cryogenically frozen sample. The sample can define a first surface and a second surface opposite the first surface. The system can also include control circuitry operatively coupled to the charged particle source and the sample stage, and one or more machine-readable storage media operatively coupled to the control circuitry. The storage media can store executable instructions that, when executed, cause the system to perform operations. The operations can include operations of the method of the first aspect in one or more embodiments.

[0014] In a third aspect, one or more machine-readable storage media store executable instructions that, when executed by a machine, cause the machine to perform operations including operations of the method of the first aspect in one or more embodiments and / or operations of the second aspect in one or more embodiments. [Brief explanation of the drawings]

[0015] The foregoing aspects and many of the attendant advantages of the present disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which:

[0016] [Figure 1] FIG. 1 is a schematic diagram illustrating an exemplary dual beam system, according to some embodiments of the present disclosure.

[0017] [Figure 2A] FIG. 1 is a perspective view of an embodiment of a multi-axis stage, according to some embodiments of the present disclosure.

[0018] [Figure 2B] FIG. 10 is a perspective view of another embodiment of a multi-axis stage, according to some embodiments of the present disclosure.

[0019] [Figure 3] FIG. 1 is a schematic diagram of a thermal control system according to some embodiments of the present disclosure.

[0020] [Figure 4A] FIG. 1 is a schematic diagram illustrating components of a vitrification holder used as part of a process for preparing HPF samples, according to some embodiments of the present disclosure.

[0021] [Figure 4B] FIG. 10 is a schematic diagram illustrating components for holding a sample grid as part of the HPF process, according to some embodiments of the present disclosure.

[0022] [Figure 5]FIG. 1 is a schematic block flow diagram illustrating a process for preparing lamellae from relatively thick samples, according to some embodiments of the present disclosure.

[0023] [Figure 6] 1A-1C are schematic diagrams illustrating an exemplary process for preparing lamellae from relatively thick samples, according to some embodiments of the present disclosure.

[0024] [Figure 7A] 1A-1D are schematic diagrams illustrating an exemplary process for preparing one or more lamellae from a relatively thick sample, according to some embodiments of the present disclosure. [Figure 7B] 1A-1D are schematic diagrams illustrating an exemplary process for preparing one or more lamellae from a relatively thick sample, according to some embodiments of the present disclosure.

[0025] [Figure 8] 1A-1C are schematic diagrams illustrating the arrangement of features for preparing multiple lamellae in a sample, according to some embodiments of the present disclosure.

[0026] [Figure 9] FIG. 1 is a schematic diagram showing a top view of a sample prepared for milling multiple lamellae, according to some embodiments of the present disclosure.

[0027] [Figure 10] FIG. 1 is a schematic diagram showing a top view of a sample prepared for milling multiple lamellae, according to some embodiments of the present disclosure.

[0028] [Figure 11] 1 is an electron micrograph showing multiple lamellae prepared in accordance with the present disclosure and prepared using a scanning electron microscope to generate secondary electron signals.

[0029] [Figure 12] 1 is an electron micrograph showing multiple lamellae prepared in accordance with the present disclosure and prepared using a focused ion beam to generate a secondary electron signal.

[0030] In the drawings, where appropriate, not every element is labeled to reduce clutter in the drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles being described. DETAILED DESCRIPTION OF THE INVENTION

[0031] While exemplary embodiments have been shown and described, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Embodiments of the present disclosure include systems, methods, algorithms, and non-transitory media having computer-readable instructions stored thereon for preparing a sample for inspection in a charged particle beam system.

[0032] While the following detailed description focuses on an embodiment of focused ion beam (FIB) preparation of cryogenic samples prepared with high-pressure freezing (HPF) to prepare lamellar samples for examination in a transmission electron microscope (TEM), it is contemplated that the described techniques can be used to improve additional and / or alternative instrument systems, sample types, and preparation techniques. In one illustrative example, the techniques described herein can be applied to multilayer semiconductor samples as an approach to precisely isolate specific layers within a sample, thereby enabling improved imaging resolution and microanalysis. Furthermore, the techniques described herein also address known challenges with thick samples, such as samples with preferred orientation, low throughput when milling small and / or low-concentration samples, and samples with poor distribution on grid squares.

[0033] 1 is a schematic diagram illustrating an exemplary dual beam system 100 according to some embodiments of the present disclosure. The exemplary system 100 includes an electron source 105, an electron beam column 107, an ion source 110, a focused ion beam (“FIB”) column 111, a gas injection system (“GIS”) 115, a vacuum chamber 120, and a sample stage 125. The electron beam column 107 is shown as a scanning electron microscope (SEM) column, such that the exemplary system 100 corresponds to a dual beam FIB-SEM system. The electron beam column 107, the FIB column 111, and the GIS 115 are shown operatively coupled to the vacuum chamber 120, with the electron beam column 107 defining a first axis A and the FIB column 111 defining a second beam axis B. Axes A and B are shown converging on a region of sample 130 toward which GIS 115 is oriented and configured to direct a gas flow containing precursors into the vacuum chamber. Advantageously, axes A and B can be oriented at different locations, but the convergence allows the SEM system to image the region of the sample being processed with the FIB.

[0034] The electron source 105 may include one or more emitters configured to generate free electrons and direct them to the electron beam column 107. The emitters may include thermionic emitters, Schottky emitters, field emission emitters, or a combination thereof, and may be operably coupled to a power system configured to apply a high voltage (e.g., on the order of several kilovolts to several hundred kilovolts) to an emitting region of the emitter material. For example, the electron source 105 may include a lanthanum hexaboride (LaB6) emitter crystal to which a high potential is applied to induce the emission of electrons from the tip of the emitter crystal. In this manner, an electron beam may be directed to the electron beam column 107.

[0035] The electron beam 107 includes electromagnetic optics (e.g., electrostatic lenses, electromagnetic lenses, monochromators, aberration correctors, etc.) and apertures configured to shape, focus, defocus, narrow, and / or direct the electron beam such that the beam is focused onto the sample 130 according to a set of operating parameters. The operating parameters may include beam current, beam energy (e.g., volts, electron-volts, etc.), a magnification parameter, a scan pattern, a dwell time, and / or one or more pulse parameters. In this manner, the exemplary system 100 functions as an SEM to image a portion of the sample 130 and / or may be used for electron-beam-assisted deposition of materials onto the sample 130 (e.g., in conjunction with the GIS 115) or other sample modification.

[0036] The ion source 110 may include one or more components configured to generate an ion beam and direct the ions into the FIB column 111. Generally, the ions may include metal ions and / or non-metal ions (e.g., noble gases, halogens, oxygen, or nitrogen). As such, the ion source 110 may include a plasma source (e.g., an inductively coupled plasma source) and / or a metal ion source (e.g., a liquid metal ion source). In the context of the present disclosure, atomic and / or molecular gases, as well as mixtures thereof, may function as plasma precursor gases from which a current of ions can be extracted.

[0037] Similar to the electron beam column 107, the FIB column 111 can include electromagnetic optics (e.g., electrostatic lenses, electromagnetic lenses, monochromators, aberration correctors, etc.) and apertures configured to shape, focus, defocus, narrow, and / or direct the ion beam such that the beam is focused onto the sample 130 according to a set of operating parameters. The operating parameters can include beam current, beam energy (e.g., volts, electron-volts, etc.), a magnification parameter, a scan pattern, a dwell time, and / or one or more pulse parameters. In this manner, the exemplary system 100 can function as a FIB used to modify portions of the sample 130 and / or (e.g., in conjunction with the GIS 115) for ion-beam-assisted removal of material from and / or deposition of material onto the sample 130.

[0038] Similar to the energies described with respect to the electron beam above, the ion beam energy can be selected (e.g., by a user, by a user-initiated algorithm, and / or automatically without user intervention). In some embodiments, additional and / or alternative precursor decomposition mechanisms (e.g., surface activation and / or secondary electron re-emission) can be used as precursor decomposition mechanisms, thereby allowing the ion beam energy to be determined based at least in part on the relationship between beam energy, sample material properties, and the energy characteristics of the precursor deposition reaction mechanism. Advantageously, ion beam-induced deposition can provide relatively high yields compared to electron beam-induced deposition, based at least in part on the combined effects of multiple energy transfer pathways.

[0039] The GIS 115 includes components that both enable the GIS 115 to generate a gas flow containing a precursor and direct the gas flow into the vacuum chamber. The components of the GIS 115 can include a carrier gas inlet, a nozzle 119, and a conduit fluidly connecting the nozzle 119 to a precursor reservoir 117. The precursor reservoir 117 can include a substantially non-reactive container (e.g., a ceramic crucible, a PTFE enclosure, a non-reactive metal or alloy, etc.) at least partially exposed to the conduit. In this manner, vapor generated from a precursor disposed in the precursor reservoir 117 can be directed into the vacuum chamber toward the nozzle (e.g., by pressure-driven flow induced by a pressure gradient relative to the vacuum of the vacuum chamber). In some embodiments, the GIS 115 includes a carrier gas inlet fluidly connected to the nozzle 119 via a conduit. In this manner, the precursor can be entrained in the carrier gas flow and directed into the vacuum chamber toward the nozzle. Additionally and / or alternatively, the precursors may comprise gases at standard conditions and may be introduced into the GIS 115 via gas inlets provided as part of the GIS 115 .

[0040] The operation of one or more components of the exemplary system 100 can be coordinated by control circuitry according to machine-executable instructions (e.g., software, firmware, etc.), which can be stored on a machine-readable storage medium and / or received from an external system via wired and / or wireless communication techniques (e.g., via a WiFi or Bluetooth link). As such, the components of the exemplary system 100 can be automated (e.g., operate without human intervention), pseudo-automated (operate with limited human intervention to initiate operations, analyze outputs, verify, etc.), or manually operated (e.g., where individual operations of the exemplary system 100 are performed and / or coordinated by a human user). In one illustrative example, the sample stage 125 can be mechanically coupled to an automated stage control 127 that enables the sample 130 to be reversibly tilted relative to beam axes A and B such that the surface of the sample is oriented at a specific angle relative to a given beam axis during operation of a corresponding charged particle beam source. In this manner, operation of a given beam source can be coordinated with operation of the stage control 127. Aspects of tilting operation are described in more detail with reference to FIG. 2 .

[0041] In some embodiments, the vacuum chamber 120 and / or other components of the exemplary system 100 (e.g., the sample stage 125, the stage control 127, etc.) can be configured for use with cryogenic samples. As such, the sample stage 125 can include heat removal elements such as a cooling loop, a high thermal conductivity sample holder, and one or more modifications to the sample stage to facilitate the introduction of the cryogenic sample into a vacuum environment. Aspects of cryogenic sample stage embodiments are described in more detail with reference to FIG. 3.

[0042] Some embodiments of the present disclosure omit one or more components of the exemplary system 100. For example, one or more of the sources 105 and 110 and / or columns 107 and 111 may be omitted. In one illustrative example, a single-beam FIB system may be configured to perform the operations for generating an ion beam. Similarly, multi-beam FIB systems other than dual-beam FIB-SEMs (e.g., FIB-laser systems in which two or more beam axes are not focused onto a predetermined region of the sample 130) may include a charged particle source of the present disclosure.

[0043] 2A is a perspective view of one embodiment of a multi-axis stage 200, according to some embodiments of the present disclosure. The multi-axis stage 200 includes a circular base 205 that supports a holder stand 210. Attached to the stand 210 is a cryogenic sample holder 215. The cryogenic sample holder 215 is removably coupled to the stand 210 and configured to hold a plurality of sample grids (not shown) oriented at an angle relative to the circular base 205.

[0044] Circular base 205 includes a rotational actuator configured to rotate holder 210 and circumferentially coupled cooling element 220 about a first rotational axis 225. Movement of the actuator thus results in rotational movement of the sample grid within sample holder stand 210 through 360 degrees of movement around base 205 of multi-axis stage 200. This allows the sample to be rotated relative to beam axes A and B of electron beam source 230 and ion beam source 235.

[0045] The stage 200 is mechanically coupled to a stage motion actuator that enables a second rotation axis 240. The second rotation axis 240 can be aligned with a plane defined by the beam axes A and B, thereby allowing the combination of rotations along the first rotation axis 225 and the second rotation axis 240 to define any angle of incidence of the charged particle beam onto the upper or lower surface of the sample grid disposed on the holder 215. As described with reference to the process of Figures 6-7, the stage 200 can be oriented such that the beam axis B is oriented toward the underside of the sample grid or the upper surface of the sample grid at an angle of incidence between about 0 degrees and about 90 degrees.

[0046] Cooling element 215 is supplied with liquid coolant via conduit 245, which is in thermal contact with cooling element 215. The liquid coolant may be or include a liquefied gas, such as liquid nitrogen, liquid helium, or a refrigerated liquid coolant formulated to maintain a liquid phase at cryogenic temperatures. The liquid coolant is delivered to cooling element 215 via coolant loop 250, which is fed through a vacuum chamber of a charged particle beam system (e.g., system 100 of FIG. 1). The inlet and outlet lines of coolant loop 250 are provided with sufficient clearance to span the full range of motion of stage 200.

[0047] FIG. 2B is a perspective view of another embodiment of a multi-axis stage 270, according to some embodiments of the present disclosure. The multi-axis stage 270 is configured to operate as part of a multi-beam cryogenic sample preparation system, which is an example of the multi-beam system 100 of FIG. 1 . The cryogenic sample preparation system of FIG. 2B includes a focused ion beam (FIB) source 280, an electron beam (SEM) source 285, and a light source 290. The stage 270 is configured to translate along a first axis 271 and rotate about a second axis 273. In this manner, a sample 275 held by the sample stage 270 can be oriented with respect to a first beam axis A and a second beam axis B. The stage 270 includes components for maintaining the sample 275 in a cryogenic state under vacuum and / or irradiation by charged particles from sources 280 and 285. For example, the stage 270 may include a relatively high thermal mass capable of removing heat from the sample 275 either by containing a coolant and / or by thermally conductive coupling with a reservoir of cryogenic material (e.g., liquid nitrogen, liquid helium, etc.).

[0048] Stage 270 may be configured to hold sample 275 using opposing adjustable grips to provide linear clearance to a relatively low angle of incidence relative to second axis B on both the top surface of sample 275 and the bottom surface of sample 275, as described in more detail with reference to the sample preparation process of this disclosure. In this manner, sample 275 may be rotated to allow ion and / or electron beams to be incident on both sides of sample 275. Light source 290 may be oriented to direct a beam of photons and / or a bright field toward sample 275. For example, light source 290 may include confocal optics 291 that allows the sample preparation process performed using sources 280 and 285 to be complemented by confocal imaging of biological structures in sample 275.

[0049] FIG. 3 is a schematic diagram of a thermal control system 300 according to some embodiments of the present disclosure. The thermal control system 300 can be used to heat or cool the multi-axis stage 200 of FIG. 2A while still allowing the stage 200 to move circularly within the imaging system. The thermal control system 300 includes a base 301 that is attached through a platform 302 via a series of connectors 304. A system of standoffs 303 can elevate the platform 302 to a desired level within the imaging system. The standoffs can be attached to the platform 302 through a series of pins 308. FIG. 3 is reproduced from U.S. Patent No. US8754384B1.

[0050] As shown, the base 301 fits within a cylindrical sleeve 310 that fits into the center of a metal ring 312. A heat transfer body 320, including heat transfer tubes 316 configured to carry a heat transfer medium, is fitted to a top surface 314 of the sleeve 310. In one embodiment, the heat transfer medium is chilled dry nitrogen or liquid nitrogen, and the temperature of the heat transfer plate 320 can be controlled by controlling the flow rate of the dry nitrogen with a flow meter (not shown) or by adding an auxiliary heat source, such as a thermal resistor. Thus, by controlling the type and amount of heat transfer medium circulating through the head transport pipe 316 (and / or by controlling the additional heat source), a user can control the resulting temperature of the heat transfer plate 320. In some embodiments, the platform 302, standoffs 303, connectors 304, and pins 308 are omitted, and the metal ring 312 and other components are adapted to couple with stage movement components, as described with reference to FIG. 2A .

[0051] A bearing ring 330 having a plurality of slots 335 is in thermal contact with the heat transfer plate 320. Each slot 335 in the bearing ring 330 is configured to hold a thermally conductive roller 340. Above the rollers 340 is a top plate 350. The top plate 350 is attached to the multi-axis stage 200 and includes a mounting bracket 360 and a centering pin 365 designed to provide heating or cooling functionality to the multi-axis stage 200. The top plate 350 can rotate about an axis driven via the base 301.

[0052] As can be envisioned, when the multi-axis stage is attached to the mounting bracket 360, the stage can rotate 360 ​​degrees on roller bearings (e.g., ball bearings or needle bearings) to maintain thermal connectivity with the heat transfer medium flowing through the pipes 316. In this embodiment, all components can be designed with high thermal conductivity. For example, the roller bearings can be made of steel, which has a conductivity of 46 W / mK. The cold stage components can be made of oxygen-free copper or other materials with high thermal conductivity (e.g., gold). In some embodiments, the shuttle receiver temperature can be reduced to -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, or even lower. In some embodiments, this device can be used to transfer heat to heat rather than cool the system by pumping heated liquid or gas through the pipes 316.

[0053] It should be noted that while the stage may be equipped with one roller bearing that provides the necessary mechanical support and degrees of freedom, the stage may also include a second roller bearing that thermally connects the stage to a fixed cooling body, such as cooled by liquid nitrogen.

[0054] An embodiment of the present disclosure includes a method for preparing planar lamellae from relatively thick high-pressure freezing (HPF) samples. The HPF preparation procedure involves a vitrification process, the components of which are illustrated in Figures 4A-4B. Figure 4A is a schematic diagram illustrating the components of a vitrification holder 400 used as part of the process for preparing HPF samples. The holder 400 includes an upper hemi-cylinder 405, a middle plate 410, and a lower hemi-cylinder 415. The middle plate 410 defines an opening 420 that receives a sample grid 425, shown in Figure 4B. The sample grid 425 may be disposed between two holding elements 430, referred to as "planchette hats." The sample grid 425 may be cryogenically frozen at high pressure by assembling the holder 400 and placing it in an anvil cell or other holder configured to apply force to the upper and lower hemi-cylinders 405 and 415. One or more conduits 435 defined within the cylinder(s) 405 and 415 can be cooled to cryogenic temperatures by introducing a coolant (e.g., liquid nitrogen) through the holder 400 while the sample grid 425 is pressurized.

[0055] The so-called "waffle method" for lamella preparation described by Kelley, Kotaro, et al. ("Waffle Method: A general and flexible approach for improving throughput in FIB-milling." Nature Communications 13.1 (2022): 1857) is limited to thin HPF samples with characteristic thicknesses of less than approximately 30 micrometers; beyond that, lamellae typically exhibit "shadow lamella" artifacts in transmission electron microscopy. In this context, the term "shadow lamella" refers to material remaining on the "back" of the prepared lamella, visible only during TEM acquisition (e.g., such material is not visible under in situ SEM images), and is only found after transferring the sample to the TEM, invalidating the lamella as a cryo-electron tomography (cryo-ET) sample. The "back" refers to the side of the sample not exposed to the incident ion beam, as opposed to the "front" side, which is exposed to the incident ion beam.

[0056] Addressing such limitations, a system of the present disclosure (e.g., exemplary system 100 of FIG. 1 ) can include a sample stage (e.g., sample stage 200 of FIG. 2 ) with a tilt range from about 0 degrees to about 300 degrees relative to a reference angle (including fractions, interpolations, and subranges thereof). Advantageously, the wide tilt angle range enables a two-sided method for preparing lamellae from samples having thicknesses greater than about 30 micrometers, including samples thicker than 100 micrometers, as described in more detail with reference to FIGS. 5-7B . Thus, the techniques of the present disclosure are suitable for relatively thick samples having a thickness greater than about 30 micrometers in the milling direction.

[0057] FIG. 5 is a schematic block flow diagram illustrating a process 500 for preparing a lamella from a relatively thick sample, according to some embodiments of the present disclosure. One or more operations of the exemplary process 500 can be performed by a computer system in communication with additional systems, including, but not limited to, a charged particle beam system, a characterization system, a network infrastructure, a database, and a user interface device. For example, the system can include the dual beam system 100 of FIG. 1 and / or the tilt stage 200 of FIGS. 2-3 , as well as control circuitry configured to operate these systems. As such, some operations of the exemplary process 500 can be encoded on one or more machine-readable storage media electronically coupled to the control circuitry. In some embodiments, at least a subset of the operations described with reference to FIG. 5 are performed automatically (e.g., without human intervention) or pseudo-automatically (e.g., with human initiation or limited human intervention). In some embodiments, one or more operations of the process 500 are omitted, reordered, and / or repeated. In some embodiments, one or more operations are added, as described in further detail with reference to FIGS. 6-12.

[0058] Process 500 improves upon the waffle method described by Kelley et al. by eliminating the limitation to relatively thin, cryogenically frozen samples less than about 30 micrometers. As previously discussed, the waffle method introduces shadowing artifacts into the lamellae, limiting its applicability to transmission electron microscopy. In contrast, process 500 can be used to prepare samples 501 having thicknesses 503 less than about 30 micrometers, as defined relative to a top surface 507, as well as samples 501 having thicknesses 507 greater than 30 micrometers. Process 500 includes, in operation 505, milling a double trench 509 into sample 501 using an ion beam 511. Trench 509 is oriented perpendicular to top surface 507 and is formed through thickness 503 of the sample. Trench 509 further defines a portion of sample 501 that is subsequently thinned to be transparent to high-energy electrons. In operation 510, process 500 includes orienting sample 501 with respect to beam axis B by tilting the sample by angle α with respect to beam axis B, and milling a first angled trench 513 through one of the two trenches 509. In operation 515, inverting sample 501 with respect to beam axis B by tilting the sample by angle β, forms a second angled trench 517 through sample 501. Together, the two trenches 513 and 517 define two opposing angled surfaces in a central portion 519 of sample 501, from which a lamella is defined by repeatedly thinning central portion 519 in operation 520.

[0059] The magnitude of angle β, including subranges, fractions, and interpolations thereof, can be from about 90 degrees to about 270 degrees. In one illustrative example, angle β can have a magnitude that allows the bottom surface 521 of sample 501 to be oriented directly in front of the ion beam (e.g., angle β is about 180 degrees). Thus, thinning central portion 519 can include repeatedly flipping sample 501 and milling widening angled trenches 513 and 517 through top surface 511 and bottom surface 521.

[0060] In contrast to the waffle method, process 500 produces lamellae that do not exhibit shadow artifacts for samples 501 having thicknesses 503 greater than about 30 micrometers. Without being bound to a particular physical phenomenon or mechanism, the thickness limitation of the waffle method may be due, at least in part, to redeposition of milled material on the inner surface of the angled trench, which can cause the underside of the lamella formed from the central portion to contain redeposited material from the sample. Unlike the waffle method, process 500 includes milling the second angled trench 517 from the underside 521. This approach shortens the path length from the surface to the central portion 519 and limits redeposition of sputtered sample material on the underside of the central portion 519.

[0061] FIG. 6 is a schematic diagram illustrating an exemplary process 600 for preparing a lamella from a relatively thick sample, according to some embodiments of the present disclosure. One or more operations of the exemplary process 600 may be performed by a computer system in communication with additional systems, including, but not limited to, a charged particle beam system, a characterization system, a network infrastructure, a database, and a user interface device. For example, the system may include the dual beam system 100 of FIG. 1 and / or the tilt stage 200 of FIGS. 2-3 , as well as control circuitry configured to operate these systems. As such, some operations of the exemplary process 600 may be encoded on one or more machine-readable storage media electronically coupled to the control circuitry. In some embodiments, at least a subset of the operations described with reference to FIG. 6 are performed automatically (e.g., without human intervention) or pseudo-automatically (e.g., with human initiation or limited human intervention). In some embodiments, one or more operations of the process 600 are omitted, reordered, and / or repeated. In some embodiments, one or more operations are added, as described in further detail with reference to FIGS. 7A-12 .

[0062] In operation 605, the exemplary process 600 includes milling a vertical trench 609 at an angle that is substantially perpendicular to the top surface. In this context, the term “vertical” refers to a relative orientation with respect to the top surface 607, rather than an absolute coordinate system. Alternatively, operation 605 can include milling the vertical trench 609 from below through the bottom surface 608. The vertical trench 609 is formed through a thickness 603 of the sample 601, with one wall of the vertical trench 609 defining a region of interest (ROI) 611 of the sample 601 from which the lamella will be formed. In operation 610, the sample 601 is oriented with respect to the beam axis B, and a first angled trench 613 is formed partway through the thickness 603 of the sample 601. The first angled trench 613 is formed by milling sample material on the opposite side of the (ROI) 611 relative to the vertical trench 609. In operation 615, a second angled trench 617 is formed in sample 601, at least in part, by translating sample 601 relative to beam axis B and traversing vertical trench 609. In contrast to process 500, operation 615 creates a fin portion 619 that is constrained on three sides instead of two, thereby reducing the degree of deformation of the sample. In operation 620, lamellar fin 612 is formed, at least in part, by widening second angled trench 617 such that ROI 619 lies at one end of lamellar fin 621 when ROI 619 is removed from sample 601.

[0063] Advantageously, the exemplary process 600 can be used not only for samples having a characteristic thickness 603 of less than 30 micrometers, but also for thicker samples without exhibiting shadowing artifacts in TEM analysis. This is due, at least in part, to a different approach to angle milling that eliminates the geometric limitations of milling the first and second angled trenches involved in the process 500 of the present technology (e.g., the waffle method). Thus, embodiments of the present disclosure enable the preparation of lamellae from relatively thick HPF samples, which is not possible with conventional techniques. Advantageously, the methods described herein provide a valuable alternative to lift-out methods.

[0064] Embodiments of the present disclosure include a method for preparing lamellae from cryogenic samples using a plasma-focused ion beam (PFIB) system configured to accept relatively thick frozen tissue or high-pressure-freeze-prepared samples. Conventional methods, such as plunge-freezing cells on grids, are not applicable when using larger sample volumes in biological studies (e.g., tissue samples). To improve sample quality, alternative cryogenic preparation techniques, such as high-pressure freezing (HPF), are used to facilitate vitreous freezing. HPF samples exhibit a distinct sample shape characterized by a larger, continuous ice layer approximately 20–300 μm thick, suggesting a novel lamella preparation strategy.

[0065] As such, the disclosed method includes efficient, rapid, and reliable cryo-lamella preparation of samples prepared by HPF, as shown in the figure above. The stage angular range constraints limit the use of current approaches that involve patterning only the top surface of the sample. The disclosed method includes an iterative milling approach that involves patterning the bottom surface of the sample to form facets near a region of interest within the sample, and repeatedly directing an ion beam to the top surface of the sample at different positions and / or angles relative to the top surface to form a lamella that includes at least a portion of the region of interest.

[0066] This approach allows the system (e.g., a stage constraining system) to process relatively thick samples (e.g., greater than about 20 microns). Additionally, embodiments can include GIS deposition of a protective layer on the underside, which provides faster and better results. For example, the protective layer can mechanically support the sample during milling and remove heat to reduce charge buildup. As discussed above, the methods described herein allow for internal structures to be included in the lamella, which presents significant challenges for regions of interest within relatively thick tissue samples. For example, conventional approaches to preparing lamellae in thick samples limit the location of the lamella to a region near the top surface of the sample. However, the present approach involves forming facets that allow for the formation of lamellae at locations throughout the sample's volume.

[0067] 7A-7B are schematic diagrams illustrating an exemplary process 700 for forming a lamella from a relatively thick sample, according to some embodiments of the present disclosure. The exemplary process 700 is an example of the process 600, which includes additional operations to, for example, enable the formation of a lamella at an intermediate depth in the sample corresponding to the sample's internal structure. Similar to the process 600, one or more operations of the exemplary process 700 may be performed by a computer system in communication with additional systems, including, but not limited to, a charged particle beam system, a characterization system, a network infrastructure, a database, and a user interface device. For example, the system may include the dual beam system 100 of FIG. 1 and / or the tilt stage 200 of FIGS. 2-3, as well as control circuitry configured to operate these systems. As such, some operations of the exemplary process 700 may be encoded in one or more machine-readable storage media electronically coupled to the control circuitry. In some embodiments, at least a subset of the operations described with reference to FIGS. 7A-7B are performed automatically (e.g., without human intervention) or pseudo-automatically (e.g., with human initiation or limited human intervention). In some embodiments, one or more operations of process 600 are omitted, reordered, and / or repeated. In some embodiments, one or more operations are added, as described in more detail with reference to FIGS.

[0068] In operation 705, a vertical trench 709 is formed in a sample 701 that includes a region of interest (ROI) 703. The vertical trench 709 is oriented substantially perpendicular to a top surface 711 of the sample 701 and extends through a bottom surface 713 of the sample. The sample 701 may include a protective layer 707 deposited on at least a portion of the bottom surface 713. As in process 600, the vertical trench 709 is formed on one side of the ROI 703. As described with reference to process 600 of FIG. 6 , the steps of process 700 include processing the sample from the bottom surface 713 rather than proceeding through the top surface 711.

[0069] In operation 710, the sample is reoriented (e.g., by one or more angular tilts of the sample holder and / or stage) to orient the bottom surface 713 toward the beam axis B, forming a first angled trench 717 through the sample 701. This reorientation may include tilting the sample by a defined angle β relative to the top surface 711 and / or the bottom surface 713. The tilt angle is shown as having a counterclockwise, “negative” direction, but in some cases may alternatively have a clockwise, “positive” direction. The magnitude of angle β may be from about 90 degrees to about 270 degrees, including subranges, fractions, and interpolations thereof. The magnitude of angle β may be based at least in part on the angle of incidence of the ion beam used in operation 710 (e.g., the relative orientation of beam axis B with respect to the bottom surface 713).

[0070] A first angled trench 717 intersects the vertical trench 709 and further defines a facet 719 within the ROI 703. The facet 719 may be oriented at an angle γ relative to the plane of the top surface. The angle γ may have an absolute value from about 0 degrees to about 90 degrees, including fractions, subranges, and interpolations thereof. The angle γ may be defined with reference to the beam axis B, such that the orientation of the sample 701 relative to the beam axis B, which may be defined by movement of the stage and / or sample holder, can be used to define the geometry of the facet 719.

[0071] Operation 710 may include widening the first angled trench 717 by gradually milling into the ROI 703. In this manner, the lamella defined in operation 725 may include a region of the ROI 703 at any position. Widening the first angled trench 717 may include translating the sample 701 such that the angle γ relative to the plane of the top surface is maintained and / or may include scanning the beam such that the angle defined by the facet is smaller than the angle γ. A smaller value of the angle γ offers the advantage of a thinner lamella after operation 702, but increases the risk of deformation during processing.

[0072] In operation 715, process 700 includes forming a protective layer on at least a portion of facet 719 and / or top surface 711. Additional surfaces may also be protected as shown. The entire surface of facet 719 may also be protected, as may the entire top surface 711. Operation 715 may include reorienting sample 701 so that the top surface is approximately perpendicular to beam axis B. Reorienting in operation 715 may include tilting the sample through one remaining rotation (e.g., an angle equal to 2π-β) or in a counter-rotational direction by angle β to return top surface 711 to a substantially normal, frontal orientation with respect to beam axis B.

[0073] Operation 715 may also include introducing a deposition precursor (e.g., an organometallic precursor such as methylcyclopentadienyltrimethylplatinum, a dielectric precursor, tetraethylorthosilicate, etc.) into the vicinity of sample 701 and decomposing the precursor on top surface 711 and / or facet 719 using the energy of the ion beam.

[0074] In operation 720, process 700 includes reorienting sample 701 and milling facet 719 at a parallel angle to form fin 723. Reorienting sample 701 includes tilting the sample at angle α so that top surface 711 is oriented face-on with respect to the incident ion beam. The magnitude of angle α can be from about 0 degrees to about 90 degrees, including subranges, fractions, and interpolations thereof. In some embodiments, angle α is defined as the complement angle to angle β used in operation 710. In this manner, facet 719 can be directed toward the front of the ion beam, with the surface of facet 719 substantially perpendicular to beam axis B.

[0075] The angle α can be carefully selected to reduce the incidence and / or likelihood of a milling artifact known as a "shark fin" resulting from perpendicular redeposition growth of sputtered sample material. Shark fin-type artifacts are more likely to occur when milling at lower angles of incidence, due at least in part to the relatively higher likelihood of sample material being removed at lower angles relative to the surface being patterned. Advantageously, the patterning techniques described herein reduce or substantially eliminate such redeposition, limiting the likelihood of resulting shark fin-type artifacts. Furthermore, the dual lamella approach shown in the electron micrographs and schematic diagrams below allows for the formation of multiple lamellae on a sample in a parallel configuration, based at least in part on directing the ion beam toward different locations on the facet 719 and repeating the operations of the disclosed process. Advantageously, the disclosed protocol can be applied to a wide range of HPF tissue samples (e.g., samples prepared with a plunge-jet-less assembly in various configurations).

[0076] The surface of facet 719 can be oriented at an angle between about 0 degrees and about 90 degrees, including subranges, fractions, and interpolations, with respect to beam axis B. In one illustrative example, sample 701 can be oriented with respect to beam axis B such that the ion beam is incident on facet 719 at an angle between 0 degrees and α. Advantageously, an angle of incidence relatively lower than normal incidence improves the quality of the produced lamella by reducing heating and associated amorphization of fin 723 during parallel milling.

[0077] The parallel milling in operation 720 involves translating the sample 701 relative to the beam axis B to repeatedly mill the top and bottom surfaces of the fin 723. In some embodiments, the ion beam is deflected to mill the top and bottom surfaces of the facet 719 in addition to, or instead of, translating the sample 701. In this manner, the fin 723 can define a trapezoidal cross-section. In contrast to process 500 described with reference to FIG. 5, the fin 723 can be attached to three sides of the sample 701 material. In some embodiments, operation 720 includes patterning one or more stress relief features in the facet 719, as described in more detail with reference to FIGS. 8-10.

[0078] In operation 725, process 700 includes thinning fin 723 to form lamella 725 from ROI 703. Lamella 725 can be thinned until the material is substantially transparent to electrons having the average energy used in transmission electron microscopy. In this manner, sample 701 can be loaded into a charged particle microscope, such as a TEM or other system, for additional microscopy and microanalysis. In some embodiments, the thinning operation can be used to reconstruct the structure of ROI 703. For example, sample 703 can include cell microstructures or other biological materials. When process 700 is performed using a multi-beam system, such as a dual-beam FIB-SEM, electron microscopy and / or microanalysis operations can be included as part of the lamella thinning.

[0079] In some embodiments, a double lamella method is implemented to improve lamella yield. Figures 8-10 are schematic diagrams illustrating aspects of the double lamella method, according to some embodiments of the present disclosure. The double lamella method involves patterning two or more lamellae in a side-by-side configuration using one or more trenches offset from the region(s) of interest along the horizontal axis labeled X.

[0080] FIG. 8 is a schematic diagram illustrating an arrangement 800 of features 805 according to some embodiments of the present disclosure. The features 805 are depicted on the surface of a facet 719 and represent portions of the surface from which material has been removed (e.g., milled or etched). In this manner, the process 700 can include multiple regions of interest 703 that can be substantially aligned and modified along a transverse diameter X. The arrangement 800 includes features 805 outside a first ROI 703-1, features 805 between the first ROI 703-1 and the second ROI 703-2, and features outside the second ROI 703-2. The features 805 can be substantially orthogonal to the transverse axis X and aligned with a second transverse axis Y. The plane defined by the axes X and Y can substantially coincide with the surface of the facet 719.

[0081] Advantageously, features 805 formed on the surface of facet 719 can relieve internal stresses within the lamella that may deform when the fin (e.g., fin 723 of FIG. 7) is thinned to a thickness that is substantially transparent to electrons. Features 805 can be linear, as shown, but can also define geometric shapes such as notches, crosses, spots, and the like, examples of which are shown in FIG. 9. However, the linear design of features 805 in exemplary arrangement 800 offers functional advantages over more complex geometries in that features 805 occupy less surface area, are less likely to induce failures in the regions between ROIs 703, are easier to manufacture, and are more amenable to automation for their manufacture.

[0082] 9 is a schematic diagram showing a view of a top surface 911 of a sample 900 prepared for milling multiple lamellae, according to some embodiments of the present disclosure. The view includes a projection of an arrangement 800 onto the top surface prior to forming facets (e.g., facet 719 in FIGS. 7 and 8 ) according to process 700 of FIG. 7 . Sample 900 includes a single vertical trench 909 formed in top surface 911 through which an ion beam can be directed to form the facets. A feature 805 is formed in the facet as described with reference to FIG. 8 .

[0083] FIG. 10 is a schematic diagram showing a view of a top surface 1011 of a sample 1000 prepared for milling multiple lamellae, according to some embodiments of the present disclosure. Similar to the diagram in FIG. 9, the diagram in FIG. 10 includes projections of features 1005 and 1010 onto the top surface 1011 prior to forming facets (e.g., facets 719 in FIGS. 7 and 8 ) according to process 700 in FIG. 7 . In contrast to sample 900 in FIG. 9 , sample 1000 includes a first vertical trench 1009-1 and a second vertical trench 1009-2 formed in surface 1011. Sample 1000 includes two regions of interest 1003. Two different types of features are defined in surface 1011: two notch-type features 1005 are formed surrounding ROI 1003, and a stress relief feature 1010 is formed between first ROI 1003-1 and second ROI 1003-2. Advantageously, the stress relief features 1010 reduce strain transfer between the first ROI 1003-1 and the second ROI 1003-2 as the facet is milled and thinned into two lamellae (e.g., lamella 727). The notch-type features 1005 are used to decouple lateral stresses in both the X and Y directions, relieve internal strain, and reduce torsional motion of the lamellae and resulting cyclic deformation (e.g., "wrinkles"), as shown in the electron micrographs generated for an exemplary parallel multi-lamellar sample prepared using process 700 of FIG. 7 and the arrangement of features 1005 and 1010 provided in FIGS. 11-12.

[0084] In the preceding description, various embodiments have been described. For purposes of explanation, specific configurations and details have been set forth to provide a thorough understanding of the embodiments. However, it will be apparent to those skilled in the art that these embodiments may be practiced without the specific details. Additionally, well-known features may be omitted or simplified so as not to obscure the described embodiments. While the exemplary embodiments described herein center around cryo-EM systems, and in particular dual-beam cryo-FIB systems, these are intended as non-limiting exemplary embodiments. Embodiments of the present disclosure are not limited to such embodiments, but rather are intended to address, among other aspects, analytical instrument systems capable of processing a wide variety of material samples in a charged particle beam instrument for further analysis to determine chemical, biological, physical, structural, or other properties, including, but not limited to, chemical structure, trace element composition, and the like.

[0085] Some embodiments of the present disclosure include a system including one or more data processors and / or logic circuitry. In some embodiments, the system includes a non-transitory computer-readable storage medium including instructions that, when executed on the one or more data processors and / or logic circuitry, cause the one or more data processors and / or logic circuitry to perform some or all of one or more methods and / or some or all of one or more processes and workflows disclosed herein. Some embodiments of the present disclosure include a computer program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to cause one or more data processors and / or logic circuitry to perform some or all of one or more methods and / or some or all of one or more processes disclosed herein.

[0086] The terms and expressions which have been employed are used as terms of description rather than of limitation, and the use of such terms and expressions is not intended to exclude the features shown and described or equivalents thereof, but it is recognized that various modifications are possible within the scope of the claims. Thus, while the present disclosure includes specific embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the appended claims.

[0087] When a term is used without explicit definition, it should be understood that the ordinary meaning of the term is intended unless it has a special and / or specific meaning in the field of charged particle microscope systems or other related fields. The terms "about" or "substantially" are used to indicate a deviation from a stated characteristic, where the deviation has little or no effect on the corresponding function, characteristic, or property of the described structure. In the illustrated example, when a dimensional parameter is described as "substantially equal" to another dimensional parameter, the term "substantially" is intended to reflect that the two compared parameters may not be equal within tolerance limits, such as manufacturing tolerances or confidence intervals inherent in the operation of the system. Similarly, when a geometric parameter, such as an alignment or angular orientation, is described as "nearly" perpendicular, "substantially" perpendicular, or "substantially" parallel, the term "about" or "substantially" is intended to reflect that the alignment or angular orientation may differ from the precisely described state within tolerance limits (e.g., not exactly perpendicular). In the case of numerical values ​​such as diameter, length, and width, the term "about" can be understood to account for a deviation of up to ±10% from the stated value. For example, a dimension of "approximately 10 mm" can represent a dimension between 9 mm and 11 mm.

[0088] This specification provides exemplary embodiments and is not intended to limit the scope, applicability, or configuration of the present disclosure. Rather, the following description of exemplary embodiments will provide those skilled in the art with an enabling description for implementing various embodiments. It will be understood that various changes can be made in the function and arrangement of elements without departing from the spirit and scope as set forth in the appended claims. Specific details are given in the description to provide a thorough understanding of the embodiments. However, it will be understood that practices may be implemented without these specific details. For example, certain system components, systems, processes, and other elements of the disclosure may be shown in schematic diagram form or omitted from the figures so as not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, components, structures, and / or techniques may be shown without unnecessary detail.

Claims

1. 1. A method for preparing a sample for inspection by a charged particle beam system, the method comprising: directing an ion beam toward a first surface of a sample for a first exposure of the first surface, the sample defining the first surface and a second surface opposite the first surface; rotating the sample by an angle β relative to a beam axis B of the charged particle beam system to orient the second surface to receive the ion beam; directing the ion beam towards the second surface of the sample; rotating the sample relative to the beam axis B to orient the first surface to receive the ion beam; directing the ion beam towards the first surface of the sample for a second exposure of the first surface.

2. The method of claim 1 , further comprising forming a trench in the first surface using the ion beam.

3. 3. The method of claim 2, wherein the trench is a first trench, the method further comprising forming a second trench in the second surface substantially aligned with the first trench.

4. The method of claim 1 , wherein the angle β has a magnitude of about 180 degrees.

5. The method of claim 1 , further comprising rotating the sample by an angle α relative to the beam axis B to orient the first surface to receive the ion beam.

6. The method of claim 5 , wherein the magnitude of α is from about 10 degrees to about 60 degrees.

7. 2. The method of claim 1, wherein directing the ion beam toward the second surface comprises orienting the second surface to receive the ion beam at an angle γ relative to the second surface.

8. 8. The method of claim 7, wherein the ion beam is directed toward the second surface to define a facet oriented at substantially the angle γ relative to the first surface. method.

9. The method of claim 8 further comprising depositing a protective layer on at least a portion of the facet.

10. The sample includes a region of interest, and the method comprises: forming a fin from the facet using the ion beam; The method of claim 8 , further comprising thinning the fin to form a lamella that includes at least a portion of the region of interest.

11. The region of interest is a first region of interest and the sample includes a second region of interest, and the method comprises: forming a first lamella from the fin that includes at least a portion of the first region of interest; The method of claim 10 , further comprising: forming a second lamella from the fin that includes at least a portion of the second region of interest.

12. The method of claim 11 , further comprising forming a stress relief cut in the facet between the first region of interest and the second region of interest.

13. The method of claim 1 , wherein the sample is a biological material prepared by high pressure freezing (HPF).

14. 1. A charged particle beam system comprising: a charged particle source; and a sample stage operably coupled to a column of the charged particle beam and configured to translate and / or rotate a cryogenically frozen sample, the sample defining a first surface and a second surface opposite the first surface; a control circuit operatively coupled to the charged particle source and the sample stage; one or more machine-readable storage media operably coupled to the control circuitry, the machine-readable storage media storing executable instructions that, when executed, cause the charged particle beam system to perform the following operations: directing an ion beam toward a first surface of the sample for a first exposure of the first surface; rotating the sample by an angle β relative to a beam axis B of the charged particle beam system to orient the second surface to receive the ion beam; directing the ion beam towards the second surface of the sample; rotating the sample relative to the beam axis B to orient the first surface to receive the ion beam; directing the ion beam towards the first surface of the sample for a second exposure of the first surface.

15. The operation is forming a first trench in the first surface using the ion beam; 15. The system of claim 14, further comprising: using the ion beam to form a second trench in the second surface substantially aligned with the first trench.

16. The system of claim 14 , wherein the angle β has a magnitude of approximately 180 degrees.

17. 16. The system of claim 15, wherein the operation further comprises rotating the sample by an angle α relative to the beam axis B to orient the first surface to receive the ion beam.

18. 20. The system of claim 17, wherein the first trench extends partway through the sample and the second trench extends through the sample.

19. 15. The system of claim 14, wherein directing the ion beam toward the second surface comprises orienting the second surface to receive the ion beam at an angle γ relative to the second surface to define a facet oriented substantially at the angle γ relative to the first surface.

20. The sample includes a region of interest, and the operation comprises: forming a fin from the facet using the ion beam directed toward the first surface; 20. The system of claim 19, further comprising: using the ion beam directed toward the first surface to thin the fin to form a lamella that includes at least a portion of the region of interest.

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