Method for large area 3d analysis of samples using glancing incidence fib milling

Abstract

Methods and apparatus for large area 3D analysis of a sample using glancing incidence FIB milling are disclosed herein. An example method includes at least: milling a sample with a focused ion beam at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample and expose a surface, and after milling, imaging the exposed surface of the sample with a charged particle beam.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 923,231, filed October 18, 2019. The disclosure of the foregoing application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present invention relates generally to large area 3D analysis in charged particle microscopy, and in particular to large area 3D analysis in charged particle microscopy using glancing incidence FIB milling. BACKGROUND

[0004] Sample volume analysis using charged particle systems is performed using a variety of techniques and charged particle microscopes. However, most of these techniques include delicate and demanding sample preparation that, if not performed correctly, can provide unusable results or destroy critical sample material. For example, conventional slice-and-view techniques use a focused ion beam to mill away a slice of sample to expose a surface for imaging, which can damage the region of interest. To protect the region of interest, a protective layer is used. Additionally, aligning the edge of the region of interest is critical for subsequent FIB milling operations. If the protective layer is not present, not strong enough, and / or not aligned ideally, the FIB milling can result in removal of the desired region for imaging. Additionally, conventional slice-and-view requires time-consuming preparation steps, is limited to high energy (30 keV) FIB milling, is relatively small in volume, is precise in cut location, requires very high FIB imaging resolution, and milling artifacts (e.g., “veiling”), are common and problematic for subsequent 3D reconstruction. While there are other volume analysis techniques that are potentially better than slice-and-view, they also have their own drawbacks and difficult sample preparation requirements. Thus, there is a need for simpler and more reliable volume analysis techniques, particularly techniques that can handle large area samples. SUMMARY

[0005] Disclosed herein are methods and apparatuses for large area 3D analysis of a sample using glancing incidence FIB milling. An example method includes at least: milling a sample with a focused ion beam at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample and expose a surface, and after the milling, imaging the exposed surface of the sample with a charged particle beam.

[0006] An example apparatus for implementing the disclosed technology includes at least a focused ion beam column, an electron beam column, a stage, and a controller. The controller includes or is coupled to a non-transitory memory including code that, when executed by the controller, causes the apparatus to mill a sample with a focused ion beam at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample, and after milling, image an exposed surface of the sample with a charged particle beam. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 is an example dual beam (DB) charged particle system according to embodiments of the present disclosure.

[0008] Figure 2A is an example side view of a sample and ion beam for large area glancing incidence FIB milling according to embodiments of the present disclosure.

[0009] Figure 2B is an example plan view of a series of milling regions of a sample according to embodiments of the present disclosure.

[0010] Figure 3 is an example method 301 according to embodiments of the present disclosure.

[0011] Figure 4 is a block diagram that illustrates a computer system 419 upon which an embodiment of the application can be implemented.

[0012] Throughout the drawings, like reference numerals will be used to designate like or corresponding components. DETAILED DESCRIPTION

[0013] Embodiments of the present application are described below in the context of a dual beam charged particle microscope configured to perform glancing angle, large area milling and imaging. The disclosed technology can provide large area volume reconstruction data for different types of materials, and the type of material under investigation can determine what ion species to use and what ion milling energy to use. However, it should be understood that the methods described herein are generally applicable to a variety of different tomographic methods and apparatuses, including both cone beam systems and parallel beam systems, and are not limited to any particular apparatus type, beam type, object type, length scale, or scan trajectory.

[0014] As used in this application and in the claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Further, the term “includes” means “comprises.” Additionally, the term “coupled” does not exclude the presence of intermediate elements between the coupled items.

[0015] The systems, devices, and methods described herein should not be construed as limiting in any way. Indeed, this disclosure is directed to all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and subcombinations with one another. The disclosed systems, methods, and devices are not limited to any particular aspect or feature or combination of aspects and features, nor do the disclosed systems, methods, and devices require that any one or more specific advantages be present or problem to be solved. Any theory of operation is merely to facilitate explanation, but is not required to explain the disclosed systems, methods, and devices.

[0016] Although the operations of some of the disclosed methods are described in a particular, sequential order for convenience, it should be understood that the described process can be carried out with the operations in different orders than illustrated, unless a particular sequencing is required (e.g., by the underlying data structure). For example, in some cases, illustrated operations can be rearranged, or omitted, or carried out concurrently. Moreover, the illustrations can not show all of the ways in which a disclosed system, method, or device can be used. Additionally, the description sometimes uses terms like "produce" and "provide" to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms will vary depending on the particular implementation and are readily recognizable by one of ordinary skill in the art.

[0017] In some instances, a value, program, or device is referred to as "lowest," "best," "smallest," etc. It will be appreciated that such terms are intended to indicate that a selection can be made among many used functional alternatives, and that such selection need not be better, smaller, or otherwise preferred to other alternatives.

[0018] There are many types of charged particle beam microscopes, such as transmission electron microscopes (TEM), scanning electron microscopes (SEM), focused ion beam (FIB) microscopes, and dual beam microscopes that include both a FIB and SEM column, to name a few. While there are many different analysis and image acquisition techniques that can be performed using such microscopes, one type of analysis technique is directed to obtaining an array of 2D images taken at different locations within a volume of a sample such that a volume reconstruction of the volume of the sample can be performed. Such techniques are sometimes referred to as slice-and-view. Slice-and-view are typically implemented in dual beam (DB) systems because they have the ability to remove sample material using the FIB column and image the sample using the SEM column. While it is also possible to image the sample using the FIB column, in some cases imaging with the SEM column can provide higher resolution.

[0019] Conventional slice view techniques include many steps that require precision and make the process lengthy and delicate. For example, slice views require lengthy sample preparation steps, such as digging large amounts of sample around the region of interest, and require deposition of a protective layer on the region of interest. Landmarks are often formed near the region of interest to help with position detection for sample movement. Additionally, the volume of sample analyzed by slice view is often small, to 50-100 pm in at least one dimension. For example, a large slice view volume can be 100 x 50 x 50 pm in X, Y, Z, respectively, for a total of about 250,000 pm 3 Larger volumes are possible, but time constraints of the milling process become prohibitive. Smaller volumes can range down to a few thousand cubic microns, while high resolution runs of the target can be 1000 pm 3 or less. Furthermore, slice thickness can be limited to 5 nm depending on the FIB column and source used, but is more typically about 10 nm. Ion milling of the slice can present other problems, such as veiling and non-uniform slice thickness. All of these problems, either alone or in combination, make slice view problematic and difficult to implement effectively. Another potential limitation is that the ion beam needs to be run at the maximum possible acceleration potential (i.e., 30 keV) due to the high requirements for imaging resolution and cutting position accuracy. However, this high energy damages biological tissue and obscures the visibility of certain cellular structures. It is highly desirable to avoid this damage, especially for soft materials.

[0020] One solution to the issues discussed above can be to perform glancing angle incidence "rotational milling" on the sample. Shallow angle rotational milling can provide large area analysis, e.g., 100 pm to 1 mm in diameter, and each "rotational milling" can remove a layer as thin as 2 nm. As used herein, rotational milling refers to milling a region of a sample using a shallow milling angle relative to the sample surface (e.g., 1 to 10 degrees from glancing incidence) at many different rotational orientations. One example of this technique includes placing the sample so that the FIB incidence angle relative to the sample surface is close to glancing. A short FIB exposure (typically a few seconds at a beam current of a few nA to 2.5 uA) is made on the desired region. The stage is rotated a fixed angle, typically in the range of 10-60 degrees. In some embodiments, for example, the stage is rotated 72 degrees so that five sites are milled to cover 360° of the surface. Milling five sites, for example, can reduce surface texturing artifacts. Typically, this stage rotation process is repeated until a complete 360 degree rotation of the sample has been achieved. In some cases, multiple rotations per milling cycle are preferred. As a result, ion flux is delivered to the sample from several different azimuthal directions, which greatly reduces milling artifacts (cording) compared to traditional top-down cross-sectional milling. The milling of one complete rotation constitutes a "slice." Alternatively, a slice can be defined by the frequency at which milling is interrupted to perform SEM imaging. Thus, a slice is typically (but not always) defined by 1 complete rotation. After each slice, the sample is imaged (e.g., by SEM imaging) of one or more regions of interest (ROIs) within the milled region.

[0021] In some embodiments, one or more ROIs can be imaged at high resolution, while the surrounding region within the milled region is imaged at lower resolution. Of course, the entire milled region can also be imaged at high resolution. As additional slices are removed and additional exposed surfaces are imaged, the number and location of tracked ROIs can change, with some ROIs possibly disappearing due to removal (or due to extending beyond the milled region), and additional ROIs possibly appearing due to exposure. As additional slices are removed and images are acquired, image recognition techniques and software can be enabled to track the various ROIs.

[0022] In this technique, the conventional fiducial points associated with the ROI can not be as useful for at least two reasons. First, because the total milled area is typically large, small fiducial points will lack visibility and will not facilitate alignment. Additionally, unless the fiducial points are placed along the perimeter of the milled area, they can be milled away, which can make them less useful. Subsequent localization accuracy in the ROI can be poor because the fiducial points are too small to be imaged with sufficient resolution. To overcome this problem, the ROI is localized using a pattern matching algorithm, and the image of the individual ROI is stored digitally and used as a reference image for subsequent slice images. As long as the thickness of the slices is relatively small, the sample structure details change little between each slice, so it is sufficient to use the image of the nth slice to match the n+1 slice pattern. However, in some embodiments, local fiducial markers can be formed so that there are reference locations for reconstructing an array of 2D slices. Due to the limitations discussed above, such local fiducials can only be useful in the image of a particular ROI (or possibly a cluster of closely spaced ROIs), but they can be locally useful in cases where the target features within the ROI are positioned at an angle relative to the sample normal. In such embodiments, the image registration technique can not be able to distinguish between a tilted feature and a stage drift, and the placement of local fiducial points can be helpful for referencing. Additionally, local fiducial points can also be used as an internal way to monitor slice thickness. For this embodiment, the local fiducial points can be designed in such a way that removing a certain thickness of the sample surface results in a predictable change in the measurable appearance of the local fiducial points.

[0023] In addition to the technical aspects discussed above (e.g. grazing angle, large area milling), the technique can also include variations in the milling parameters, such as ion species based on sample type, number of milling sites per revolution, ion dose per site, and milling energy. For example, in the case of biological samples, it is preferred to use O + The ion operated plasma FIB system, resulting in a mixture of molecular and atomic oxygen ions, is typically 12 keV or lower. However, if Xenon (Xe+) is used for biological samples, the ion energy should be kept below 5 keV. For other sample types, such as metal alloys or mineralogical samples, Argon (Ar+) or Xe+ can be preferred, and the range of ion energies can be large, e.g. 2-30 keV, depending on the characteristics of the individual sample. The metal and metal alloy category can include manufactured structures, such as battery electrodes, flexible displays, and integrated circuits, to name a few.

[0024] Figure 1is an example dual-beam (DB) charged particle system 100 according to embodiments of the present disclosure. The DB system 100 includes both a focused ion beam (FIB) column and an electron column such that ion processing and / or imaging along with electron imaging can be performed. In some embodiments, a combination of ion beam processing (e.g., milling) and electron imaging can be performed in a recursive technique on a large area of a sample (e.g., a 1 mm diameter region) such that a large volume of a sample is imaged. Such images can then be used for volume reconstruction of at least a portion of the sample within the large area. While examples of suitable hardware are provided below, the present invention is not limited to implementation with any particular type of hardware.

[0025] A scanning electron microscope 141 and a power supply and control unit 145 are provided with the dual-beam system 100. An electron beam 143 is emitted from a cathode 152 by applying a voltage between the cathode 152 and an anode 154. The electron beam 143 is focused to a fine point by means of a condenser lens 156 and an objective lens 158. The electron beam 143 is scanned two-dimensionally over the specimen by means of deflection coils 160. The operation of the condenser lens 156, the objective lens 158 and the deflection coils 160 is controlled by the power supply and control unit 145.

[0026] The electron beam 143 can be focused onto a sample 122 that is located on a movable X-Y stage 125 within the lower chamber 126. When the electrons in the electron beam hit the substrate 122, secondary electrons are emitted. These secondary electrons are detected by a secondary electron detector 140 as discussed below. As discussed above, a STEM detector 162 located below the TEM sample holder 124 and the stage 125 can collect electrons transmitted through a sample mounted on the TEM sample holder.

[0027] The dual-beam system 100 also includes a focused ion beam (FIB) system 111 that contains a vacuum chamber with an upper neck portion 112 in which an ion source 114 and a focusing column 116 including an extraction electrode and electrostatic optics are placed. In some embodiments, the axis of the focusing column 116 is tilted 52 degrees from the axis of the electron column. Of course, other tilt angles between the FIB and SEM columns are possible. The ion column 112 includes an ion source 114, an extraction electrode 115, focusing elements 117, deflection elements 120, and a focused ion beam 118. The focused ion beam 118 passes from the ion source 114 through the focusing column 116 and between electrostatic deflection devices indicated schematically at 120 toward a sample 122, which can be, for example, a biological sample, a semiconductor sample, a metal or metal alloy sample, or a mineralogical sample, located on a movable X-Y stage 125 within the lower chamber 126.

[0028] The stage 125 can preferably move in the horizontal plane (X and Y axes) and vertically (Z axis). The stage 125 can also tilt about sixty (60) degrees and rotate about the Z axis. In some embodiments, a negative stage tilt can be used to achieve a desired glancing FIB milling incidence angle. Of course, both positive and negative tilts are contemplated herein. The desired tilt range for an instrument that implements rotational milling is typically -38° to 60°, where 0 degrees is the "untilted" SEM normal orientation. In some embodiments, a positive tilt of 52° is orthogonal to the ion beam. In some embodiments, the stage 125 can be cooled to cryogenic temperatures by being coupled to a cold finger (not shown) that is provided with or in contact with a liquid nitrogen source. By cooling the stage 125 to cryogenic temperatures, a biological sample that is cooled to cryogenic temperatures and possibly vitrified can be milled and imaged, as discussed herein.

[0029] The neck portion 112 is evacuated with an ion pump 168. The chamber 126 is evacuated with a turbomolecular and mechanical pumping system 130 under the control of a vacuum controller 132. The vacuum system provides a vacuum of between about 1 x 10"7Torr and 5 x 10"4Torr within the chamber 126. If an etch assist gas, etch delay gas, or deposition precursor gas is used, the chamber background pressure can be increased, typically to about 1 x 10"5Torr.

[0030] A high voltage power supply provides the appropriate acceleration voltage to the electrodes in the column 116 for energizing and focusing the ion beam 118. As it strikes the substrate 122, physical ejection of material is sputtered from the sample. Alternatively, the ion beam 118 can dissociate a precursor gas to cause material to be deposited.

[0031] A high voltage power supply 134 is connected to the liquid metal ion source 114 and to the appropriate electrodes in the ion beam column 116 for forming and directing the ion beam 118 at about 1 keV to 60 keV toward the sample. Deflection controllers and amplifiers 136 operating according to a prescribed pattern provided by a pattern generator 138 are coupled to the deflection plates 120, whereby the ion beam 118 can be manually or automatically controlled to trace out a corresponding pattern on the upper surface of the substrate 122. In some systems, the deflection plates are placed before the final lens, as is well known in the art. A beam blanking electrode (not shown) within the ion beam column 116 causes the ion beam 118 to impinge on a blanking aperture (not shown) instead of the substrate 122 when a blanking controller (not shown) applies a blanking voltage to the blanking electrode.

[0032] In some embodiments, the ion source 114 is a liquid metal ion source that typically provides a beam of metal ions of gallium. The source is typically capable of focusing into a beam that is a sub-tenth of a micron wide at the substrate 122 to alter the substrate 122 by ion milling, enhanced etching, material deposition, or for imaging the substrate 122. However, in other embodiments, the ion source 114 is a plasma-based ion source, such as an inductively coupled plasma source or a radio frequency ion source, and is also capable of providing different ion species, such as oxygen, argon, xenon, and nitrogen, to name a few. In such embodiments, the plasma gas is switched to provide the desired ion species. As disclosed herein, the ion species used when operating the system 100 can depend on the sample type. For example, if the sample is a biological sample, oxygen or xenon can be the desired ion species. On the other hand, if the sample is a metal, metal alloy, or mineral, argon or xenon can be the desired ion species. As disclosed herein, the selection of the ion species based on the sample type facilitates efficient and optimal processing of the sample to mitigate the issues discussed above, such as surface texturing.

[0033] A charged particle detector 140 for detecting secondary ions or electron emission, such as an Everhart Thornley or multi-channel plate, is connected to a video circuit 142 that provides drive signals to a video monitor 144 and receives deflection signals from the system controller 119. The location of the charged particle detector 140 within the lower chamber 126 can vary in different embodiments. For example, the charged particle detector 140 can be coaxial with the ion beam and include an aperture that allows the ion beam to pass through. In other embodiments, the secondary particles can be collected by a final lens and then diverted off axis for collection.

[0034] A micro manipulator 147 can precisely move objects within the vacuum chamber. The micro manipulator 147 can include a precision motor 148 located outside the vacuum chamber to provide X, Y, Z, and theta control of a portion 149 located within the vacuum chamber. The micro manipulator 147 can be equipped with different end effectors for manipulating small objects. In the embodiments described herein, the end effector is a fine probe 150.

[0035] A gas delivery system 146 extends into the lower chamber 126 for introducing and directing gaseous vapors to the substrate 122. For example, iodine can be delivered to enhance etching, or metal organic compounds can be delivered to cause metal deposition.

[0036] The system controller 119 controls the operation of the various parts of the dual beam system 110. Through the system controller 119, a user can cause the ion beam 118 or electron beam 143 to scan in a desired manner by inputting commands into a conventional user interface (not shown). Alternatively, the system controller 119 can control the dual beam system 110 according to programmed instructions stored in memory 121. In some embodiments, the dual beam system 110 incorporates image recognition software to automatically identify regions of interest, after which the system can extract the sample manually or automatically according to the present invention. For example, the system can automatically locate a desired feature on the sample.

[0037] In operation, the system 100 performs one or more "rotary milling" processes on the sample 122 and after each rotary milling process, the exposed layer is imaged. Rotary milling involves milling the sample 122 at a glancing angle and at a plurality of rotational orientations. For example, the stage 125 is tilted so that the ion beam 118 is at 1° to 10° from the surface of the sample 122, and then the surface is milled over a desired area (the desired area can be 100 μιη to 1 mm, and can also include the entire field of view at a given magnification) on the desired area. For example, after milling on the desired area, the sample 122 is rotated by 72°, and then the sample 122 is again milled at the same glancing angle and over the same size area. Of course, other rotational angles within 360° can be used. This milling and rotating process can be repeated a desired number of times, such as 2, 3, 4, 5, 6, or more, and once a full 360° rotation has occurred (if multiple rotations are desired), the sample 122 is imaged with the electron beam 143. More specifically, after the full milling, the exposed surface of the sample 122 is imaged. The milling after a full 360° has occurred can be referred to herein as a "slice" of the sample. In some embodiments, the sample 122 can be repositioned in x, y, z, and / or tilt angle to acquire an image, and then repositioned to the desired glancing angle to mill another slice of the sample 122.

[0038] For milling, the ion species used and the energy of the ion beam 118 can be based on the type of sample being milled and imaged. For example, O2+ can be used to mill biological samples at 12 keV or less, or Xe+ at less than 5 keV. If the sample is a metal or mineral, for example, the ion species can be Ar+ or Xe+ at an energy of 2 to 30 keV. Regardless of the ion species, sample, and ion beam energy, the sample can also be cooled to cryogenic temperatures during the milling imaging process. Cryogenic cooling can be particularly useful in studying biological samples, where some samples can have been vitrified prior to loading into the system 100 to preserve their structure. By implementing a "rotational milling" process, large areas of the sample 122 can be repeatedly imaged such that a certain number of images can form a 3D reconstruction of the sample. Additionally, the use of the disclosed rotational milling process reduces the need for depositing a protective layer, pre-milling a large volume around the ROI, and depositing fiducials, which provides a direct and reliable 3D analysis technique.

[0039] Figure 2A is an example side view of a sample 222 and ion beam 218 for large area, glancing incidence FIB milling according to embodiments of the present disclosure. Figure 2A The illustration of FIG. 1 is an example of FIB milling that can be implemented on a DB system, such as the system 100. The illustration shows a sample 222 being milled by an ion beam 218 at an angle Θ. Milling occurs on a region 223 of the sample 222, where the region 223 is shown as a dashed area. The ion beam 218 is at an angle Θ to the surface of the sample 222, where Θ ranges from 1° to 10°. In general, Θ is defined as the glancing angle with respect to the sample 222. The sample 222 can be milled at the glancing angle from multiple different rotational orientations (e.g., 2, 3, 4, 5, 6, etc.) to remove a layer from the region 223 (see, e.g., FIG. 2). Figure 2B Based on the ion beam 218 energy, ion beam current, and angle of the ion beam 218, the layer removed can be as thin as 2 nm, but can be in the range of 2 to 10 nm, for example. Additionally, by milling the region 223 from multiple rotational orientations, the exposed surface of the region 223 can be free of defects and undesirable textures, such as veiling, providing better images and ultimately a better reconstructed 3D volume.

[0040] Figure 2Bis an example plan view of a series of milled regions 223 of a sample 222 according to embodiments of the present disclosure. The series of milled regions includes milled regions A, B, C, D, and E, all of which are performed by ion beam 218 with the sample in different rotational orientations. By rotating the sample 222 between each milling operation, the series of milled regions 223 A-E forms a generally circular region that receives ion beam milling at each rotational orientation. This generally circular region then forms a "slice" that exposes a subsurface region. The exposed surface can then be imaged. After imaging, the series of mills can be performed again to expose a subsequent surface for imaging. This process can be repeated as many times as desired to image the sample 222 to a desired depth.

[0041] Each of the milled regions A-E is milled by delivering the ion beam 218 to each pixel in the respective square. As used herein, the term "pixel" refers to a coordinate on the sample 222 within a milled region that receives the ion beam 218 for a specified amount of dwell time, such as a few to a few hundred microseconds, but the dwell time can depend on the beam current, to mill away some of the sample 222 at the coordinate. In other words, each pixel receives the ion beam 218 for the desired dwell time. It can be seen that the regions outside of the "slice" circle also receive the ion beam 218, but because those regions do not receive the ion beam at every rotational orientation, they can not have the entire thickness of the sample 222 milled away. Additionally, each milled region A-E can fill the entire field of view of a microscope at a given magnification of the ion column, such as ion column 111. In this way, a very large region can be milled at each rotational orientation.

[0042] Figure 3 is an example method 301 according to embodiments of the present disclosure. The method 301 can be implemented on a DB system, such as system 100. The method 301 can produce a series of images of a large area sample that has had a series of layers removed using a glancing angle FIB mill. Additionally, the ion species and milling energy can be adjusted depending on the sample material.

[0043] The method 301 begins at optional process block 303, which includes determining the sample material type. For example, it can be determined that the sample material type is biological, metallic, semiconductor, mineral, etc. In some embodiments, this determination can be made by a user. In other embodiments, this determination can be made automatically by the system using some other analysis technique, such as spectroscopy. For example, a spectroscopic analysis, such as using EBSD, can be performed to determine the chemical composition of the sample. The chemical composition can determine whether the sample is a metallic / alloy sample, a semiconductor sample, or a biological sample, which can determine the milling parameters and ion species.

[0044] Process block 303 can be followed by optional process block 305, which includes setting milling parameters based on the sample material. The milling parameters can include, for example, ion species, milling energy, and ion beam current. Generally, the sample material type dictates the milling parameters to achieve a high surface quality milling. For example, if the sample is biological, the selected ion species can be oxygen or xenon. If oxygen is selected, the ion beam energy can be set to 12 keV or less. If xenon is selected, the ion beam energy can be set to less than 5 keV. For example, for a metal or mineral type sample, the ion species can be argon or xenon, and both can be delivered at a range of energies, such as 2 keV to 30 keV.

[0045] Process block 305 can be followed by process block 307, which includes milling the sample at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample. Removing the layer exposes a surface of the sample. The shallow angle can be, for example, 1 to 10 degrees, and the number of rotational orientations can be 2 to 10. Additionally, the milling can be performed stepwise, as each rotational orientation is maintained for a desired amount of milling time before the sample is rotated to a subsequent rotational orientation. Alternatively, the sample can be continuously rotated while the milling is performed for a desired amount of time.

[0046] Process block 307 can be followed by process block 309, which includes imaging the exposed surface of the sample after the milling. The imaging can be performed after all of the rotational orientations are milled or after each individual rotational orientation is milled. The image can then be stored, for example.

[0047] Optionally, after performing process block 309, method 301 can return to process block 307 to remove another layer of the sample by milling at a plurality of rotational orientations. This optional loop can be performed multiple times until the sample at a desired depth is imaged. In this way, an image of each respective surface of the sample is obtained.

[0048] Optionally, process block 309 can be followed by process block 311, which includes forming a 3D reconstruction of the imaged area of the sample. The images of the plurality of exposed surfaces are combined to form the 3D reconstruction.

[0049] Figure 4 FIG. 41 is a block diagram that illustrates a computer system 419 upon which an embodiment of the application can be implemented. The computing system 419 can be an instance of the system controller 119. The computer system 419 includes at least the bus or other communication mechanism for communicating information and a hardware processor, e.g., a core, 470, coupled on the bus for processing information. The hardware processor 470 can be, for example, a general purpose microprocessor. The computing system 419 can be used to implement the methods and techniques disclosed herein, such as method 301, and can also be used to obtain images and process the images with one or more filters / algorithms.

[0050] Computer system 419 also includes a main memory 421, such as a random access memory (RAM) or other dynamic storage device, coupled to bus 420 for storing information and instructions to be executed by processor 470. Main memory 421 also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 470. Such instructions, when stored in non-transitory storage media accessible to processor 470, render computer system 419 into a special-purpose machine that operates to perform the operations specified in the instructions.

[0051] Computer system 419 further includes a read only memory (ROM) 472 or other static storage device coupled to bus 420 for storing static information and instructions for processor 470. A storage device 474, such as a magnetic disk or optical disk, is provided and coupled to bus 420 for storing information and instructions.

[0052] Computer system 419 can be coupled via bus 420 to a display, such as a cathode ray tube (CRT), for displaying information to a computer user. An input device, including alphanumeric and other keys, is coupled to bus 420 for communicating information and command selections to processor 470. Another type of user input device is cursor

[0053] Computer system 419 can implement the techniques described herein using customized hard-wired logic, one or more ASICs or FPGAs, firmware and / or program logic which in combination with the computer system causes or programs computer system 419 to be a special-purpose machine. According to one embodiment, the techniques herein are performed by computer system 419 in response to processor 470 executing one or more sequences of instructions contained in main memory 421. Such instructions can be read into main memory 421 from another storage medium, such as storage device 474. Execution of the sequences of instructions contained in main memory 421 causes processor 470 to perform the process steps described herein. In alternative embodiments, hard-wired circuitry can be used in place of or in combination with software instructions.

[0054] The term "storage media" as used herein refers to any non-transitory media that store data and / or instructions that cause a machine to operate in a specific fashion. Such storage media can comprise non-volatile media and / or volatile media. Non-volatile media includes, for example, optical disks or magnetic disks, such as storage device 736. Volatile media includes dynamic memory, such as main memory 732. Common forms of storage media include, for example, a floppy disk, a flexible disk, hard disk, solid state drive, magnetic tape, or any other magnetic data storage medium, a CD-ROM, any other optical data storage medium, any physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, NVRAM, any other memory chip or cartridge, content-addressable memory, and a tri-state content- addressable memory (TCAM).

[0055] Storage media are tangible and non-transitory. Storage media differs from transmission media, which are channels for propagating signals, such as electrical, electromagnetic or optical signals. Transmission media can contain storage media. Common forms of physical and optical transmission media include air, coaxial cable, twisted pair wire, copper wire, fiber optics, and carrier waves that travel through the air, cables, and / or wires, including wires that form a bus.

[0056] Computer system 419 also includes a communication interface 476 coupled to bus 420. Communication interface 476 provides a two-way data communication coupling to, for example, a network link 478 that is connected to a local network. As another example, communication interface 476 can be a local area network (LAN) card to provide data communication connection to a compatible LAN. Wireless links can also be implemented. In any such implementation, communication interface 476 sends and receives electrical, electromagnetic or optical signals that carry digital data streams representing various types of information.

[0057] Computer system 419 can send messages and receive data, including program code, through the network(s), network link 478 and communication interface 476. In the Internet example, a server might transmit a requested code for an application program through the Internet, ISP, local network and communication interface 476. The received code can be executed by processor 470 as it is received, and / or stored in storage device 736, or other non-volatile storage for later execution.

[0058] The discussion herein to illustrate embodiments of the disclosed technology should not be considered limiting and merely provides examples of implementations. For example, different numbers of rotational orientations, ion species, ion beam energies, and currents can be implemented at various milling angles and still fall within the scope of the present disclosure. Those skilled in the art will understand how other myriad ways of implementing the disclosed technology can be implemented, which are contemplated herein and within the scope of the present disclosure.

[0059] An example method of implementing the disclosed technology includes milling a sample with a focused ion beam at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample, and after the milling, imaging an exposed surface of the sample with a charged particle beam.

[0060] The above example method, wherein each of the plurality of rotational orientations is individually milled prior to a subsequent rotational orientation of the sample being rotated to.

[0061] The above method, wherein the plurality of rotational orientations are milled while the sample is being rotated.

[0062] The above example method, wherein the sample is a biological sample.

[0063] The above example method, wherein the focused ion beam provides oxygen ions at an energy of 12 keV or less.

[0064] The above example method, wherein the focused ion beam provides xenon ions at an energy of less than 5 keV.

[0065] The above example method, wherein the sample is a metal or metal alloy.

[0066] The above example method, wherein the focused ion beam provides argon or xenon at an energy in a range of 2 to 30 keV.

[0067] The above example method, wherein the sample is a mineral.

[0068] The above example method, wherein the focused ion beam provides argon or xenon at an energy in a range of 2 to 30 keV.

[0069] The above example method, wherein the sample is held on a cryogenic stage that is cooled to a low temperature.

[0070] The above example method, wherein the sample is cooled to a low temperature while being milled and imaged.

[0071] The above example method, wherein the sample is moved during imaging to acquire an image of a region of interest.

[0072] The above example method, wherein the milling is of a region having a diameter of up to 1 mm.

[0073] The above example method, wherein the sample is milled at a shallow angle and at a plurality of rotational orientations to remove a layer of 2 nm thickness.

[0074] The above example method, wherein the shallow angle is in a range of 1 to 6 degrees from a surface of the sample.

[0075] The above example method, wherein the focused ion milling is plasma focused ion milling.

[0076] The example method above, wherein the focused ion plasma milling is capable of switching between different ion species.

[0077] The example method above, wherein the different ion species include oxygen, argon, and xenon.

[0078] The example method above, further comprising repeating the milling and imaging steps multiple times.

[0079] The example method above, further comprising identifying a sample material type.

[0080] The example method above, further comprising setting milling parameters, such as ion species, milling energy, ion beam current, and dwell time, based on the sample material type.

[0081] An example apparatus for implementing the disclosed technology comprises at least a focused ion beam column, an electron beam column, a stage, and a controller. The controller comprises or is coupled to a non-transitory memory comprising code that, when executed by the controller, causes the apparatus to mill a sample with a focused ion beam at a shallow angle and at a plurality of rotational orientations to remove a layer of the sample, and to image an exposed surface of the sample with a charged particle beam after the milling.

[0082] The example apparatus above, wherein each of the plurality of rotational orientations is individually milled before a subsequent rotational orientation of the sample is rotated to.

[0083] The example apparatus above, wherein the plurality of rotational orientations are milled while the sample is being rotated.

[0084] The example apparatus above, wherein the sample is a biological sample.

[0085] The example method above, wherein the focused ion beam provides oxygen ions at an energy of 12 keV or less.

[0086] The example apparatus above, wherein the focused ion beam provides xenon ions at an energy of less than 5 keV.

[0087] The example apparatus above, wherein the sample is a metal or metal alloy.

[0088] The example apparatus above, wherein the focused ion beam provides argon or xenon at an energy in a range of 2 to 30 keV.

[0089] The example apparatus above, wherein the sample is a mineral.

[0090] The example apparatus above, wherein the focused ion beam provides argon or xenon at an energy in a range of 2 to 30 keV.

[0091] The example apparatus above, wherein the sample is held on a cryogenic stage that is cooled to a low temperature.

[0092] The example apparatus above, wherein the sample is cooled to cryogenic temperatures while milling and imaging.

[0093] The example apparatus above, wherein the sample is moved during imaging to acquire images of regions of interest.

[0094] The example apparatus above, wherein regions up to 1 mm in diameter are milled.

[0095] The example apparatus above, wherein the sample is milled at shallow angles and at multiple rotational orientations to remove 2 nm thick layers.

[0096] The example apparatus above, wherein the shallow angles are in the range of 1 to 6 degrees from the surface of the sample.

[0097] The example apparatus above, wherein the focused ion milling is plasma focused ion milling.

[0098] The example apparatus above, wherein the plasma focused ion milling is capable of switching between different ion species.

[0099] The example apparatus above, wherein the different ion species include oxygen, argon, and xenon.

[0100] The example apparatus above, further comprising repeating the milling and imaging steps multiple times.

[0101] The example apparatus above, further comprising identifying a sample material type.

[0102] The example apparatus above, further comprising setting milling parameters such as ion species, milling energy, ion beam current, and dwell time based on the sample material type.

Claims

1. A method comprising: milling the sample with a focused ion beam at a shallow angle and at multiple rotational orientations to remove a layer of the sample; and After milling, imaging the exposed surface of the sample with a charged particle beam; Wherein the local fiducials are designed in such a way that removal of a specific thickness of the sample surface results in a predictable change in the measurable appearance of the local fiducials, and wherein one or more regions of interest are imaged at high resolution while surrounding areas within the milled region are imaged at lower resolution.

2. The method of claim 1 , wherein the rotational orientation comprises orienting the sample relative to the focused ion beam into the plurality of rotational orientations, and wherein each of the plurality of rotational orientations is individually milled before rotating the sample to a subsequent rotational orientation of the plurality of rotational orientations. The method of claim 1 , wherein the shallow angle is in the range of 1 to 6 degrees from the surface of the sample. The method of claim 1 , further comprising cooling the sample to a cryogenic temperature.

5. The method of claim 1, further comprising moving the sample during imaging to acquire images of different regions within the milled area. The method according to claim 1 , wherein the milling is performed on an area having a diameter of up to 1 mm.

7. The method of claim 1, wherein the shallow angle is in the range of 1 to 10 degrees from the surface of the sample, and wherein milling the sample at the shallow angle and at the plurality of rotational orientations removes a 2 nm thick layer.

8. The method according to claim 1, further comprising: determining the material type of the sample; and Based on the material type, set the milling parameters.

9. The method of claim 8, wherein the milling parameters include ion species, ion beam current, ion beam energy, and dwell time.

10. The method of claim 9, wherein the ion species include oxygen, argon, and xenon.

11. An apparatus comprising: a focused ion beam column coupled to provide a focused ion beam, wherein the focused ion beam is a plasma-based focused ion beam capable of switching to different ion species; an electron column connected to the electron column to provide an electron beam; a stage arranged to hold a sample, wherein the stage is at least tiltable and rotatable; and a controller coupled to or including non-transitory memory, the non-transitory memory including code that, when executed by the controller, causes the device to: orienting the stage at a shallow angle relative to the focused ion beam; milling the sample with the focused ion beam at the shallow angle and at a plurality of rotational orientations to remove a layer of the sample; and After milling, the exposed surface of the sample is imaged with the electron beam; wherein the local fiducials are designed in such a way that removing a specific thickness of the sample surface results in a predictable change in a measurable appearance of the local fiducials, and wherein one or more regions of interest are imaged at high resolution, while surrounding areas within the milled region are imaged at lower resolution.

12. The apparatus of claim 11, wherein the rotational orientation comprises orienting the sample relative to the focused ion beam into the plurality of rotational orientations, and wherein each of the plurality of rotational orientations is individually milled before rotating the sample to a subsequent rotational orientation of the plurality of rotational orientations.

13. The apparatus of claim 11, wherein the shallow angle is in the range of 1 to 6 degrees from the surface of the sample.

14. The apparatus of claim 11, wherein the stage is a cryogenic stage and the non-transitory memory includes code that, when executed by the controller, causes the apparatus to cool the sample to a cryogenic temperature.

15. The apparatus of claim 11, wherein an area up to 1 mm in diameter is milled.

16. The apparatus of claim 11, wherein the shallow angle is in the range of 1 to 10 degrees from the surface of the sample, and wherein a 2 nm thick layer is removed during the milling operation.

17. The apparatus of claim 11 , wherein the non-transitory memory comprises code that, when executed by the controller, causes the apparatus to: determining the material type of the sample; and Based on the material type, set the milling parameters.

18. The apparatus of claim 17, wherein the milling parameters include ion species, ion beam current, ion beam energy, and dwell time.

19. The apparatus of claim 18, wherein the ion species include oxygen, argon, and xenon.

20. The apparatus of claim 17, wherein the ion species is set to oxygen, and the energy of the focused ion beam is set to a maximum value of 12 keV, based on the sample being a biological sample.

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