Method and system for imaging sample

Abstract

To provide a high-quality sample image with low-dose radiations.SOLUTION: A sample is imaged by directing a charged particle beam towards the sample and forming an irradiation zone. The charged particle beam is scanned such that the irradiation zone is scanned in the sample plane in a first direction and radiations from a detection zone are detected by a detector. A first number of detecting pixels arranged along a first detector axis corresponding to the first detection axis is fewer than a second number of detecting pixels arranged along a second detector axis corresponding to the second detection axis.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This description relates generally to methods and systems for imaging a sample using a charged particle microscope, and more particularly to scanning a sample and collecting data in a charged particle microscope. Summary of the Invention

[0002] In one embodiment, a method for imaging a sample includes directing a charged particle beam towards the sample to form an illumination zone in a sample plane; scanning the illumination zone in substantially a first direction in the sample plane and detecting radiation from the detection zone using a plurality of detection pixels of a detector, where a first detector axis of the detector corresponds to a first detection axis of the detection zone and a second detector axis of the detector corresponds to a second detection axis of the detection zone, a first number of the plurality of detection pixels arranged along the first detector axis being less than a second number of the plurality of detection pixels arranged along the second detector axis, and the first detection axis being oriented at an angle of less than 45 degrees from the first direction; and reconstructing an image of the sample based on the radiation detected during the scan.

[0003] In another embodiment, a method for imaging a sample includes directing a charged particle beam at the sample to form an illumination zone in a sample plane, detecting a first radiation from the detection zone of the sample using a plurality of detection pixels of a detector arranged along a first detector axis and a second detector axis, where a first number of the plurality of detection pixels arranged along the first detector axis is less than a second number of the plurality of detection pixels arranged along the second detector axis, moving the illumination zone and the detection zone in a first direction in the sample plane, detecting a second radiation from the detection zone using the plurality of detection pixels, where a first detection axis of the detection zone corresponds to the first detector axis and a second detection axis of the detection zone corresponds to the second detector axis, and an angle between the first direction and the first detection axis is less than 45 degrees, and reconstructing an image of the sample based on the first radiation and the second radiation.

[0004] In yet another embodiment, a charged particle microscope includes a sample holder for positioning a sample in a sample plane, a charged particle column for directing a charged particle beam toward the sample plane to form an illumination zone in the sample plane, a pixelated detector for detecting radiation generated from the detection zone of the sample in response to illuminating the sample with the charged particle beam, a first detector axis of the detector corresponding to a first detection axis of the detection zone and a second detector axis of the detector corresponding to a second detection axis of the detection zone, and a controller including a non-transitory memory for storing computer readable instructions and a processor, wherein by executing the computer readable instructions in the processor, the charged particle microscope is configured to scan the illumination zone in substantially a first direction in the sample plane via the charged particle column, detect radiation from the detection zone via a plurality of pixels of the detector, wherein a first number of a plurality of detection pixels arranged along the first detector axis is less than a second number of a plurality of detection pixels arranged along the second detector axis and an angle of the first direction from the first detection axis is less than 45 degrees, and reconstruct an image of the sample based on the radiation detected during the scan.

[0005] It should be understood that the foregoing Summary is provided to introduce a selection of concepts in a simplified form that are further described in the Detailed Description. It is not intended to identify key or essential features of the claimed subject matter, the scope of which is uniquely defined by the claims that follow the Detailed Description. Moreover, the claimed subject matter is not limited to implementations that solve any disadvantages discussed above or in any part of this disclosure. [Brief description of the drawings]

[0006] [Figure 1] Illustrates an embodiment of a charged particle microscope. [Diagram 2] 1 illustrates one embodiment of collecting data from a circular sample area. [Figure 3A] 1 shows exemplary illumination zones and corresponding detection zones. [Figure 3B] 1 shows exemplary illumination zones and corresponding detection zones. [Figure 3C] 3 illustrates one exemplary arrangement of detection pixels of a detector. [Figure 3D] 4 illustrates another exemplary illumination zone and corresponding detection zone. [Figure 4A] Illustrates an exemplary data collection scheme. [Figure 4B] Illustrates an exemplary data collection scheme. [Figure 4C] Illustrates an exemplary data collection scheme. [Figure 4D] Illustrates an exemplary data collection scheme. [Figure 4E] Illustrates an exemplary data collection scheme. [Figure 4F] Illustrates an exemplary data collection scheme. [Figure 4G] Illustrates an exemplary data collection scheme. [Figure 4H] Illustrates an exemplary data collection scheme. [Diagram 5] 1 illustrates another embodiment for collecting data from a circular sample area. [Figure 6] A portion of FIG. 5 is shown. [Figure 7] 1 illustrates an exemplary method for imaging a sample.

[0007] Like reference numbers refer to corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The following description relates to a system and method for imaging a sample using a charged particle microscope. A charged particle microscope can be used to analyze the features and structure of a sample with high resolution. For radiation sensitive samples, high quality sample imaging with low dose radiation can be difficult. However, detectors with high sensitivity and low noise levels can be expensive.

[0009] As an example, transmission electron microscopy Electron microscopy (TEM) is a type of charged particle microscope that can provide high-resolution details of biological structures. One application of TEM is for single particle analysis (SPA), which can reconstruct the structure of particles such as proteins or viruses. Samples for SPA are usually highly radiosensitive, so lower doses of radiation are required during an imaging session. Direct electron detectors (DEDs) are the primary technology for low-dose imaging. However, DEDs with large fields of view, which require detectors with large arrays (i.e., with a large number of detection pixels), may be out of reach of potential users due to their high cost.

[0010] Furthermore, the process for sample preparation can be very complicated and time-consuming. For example, in SPA, the sample (e.g., particle morphology) is vitrified and imaged in holes of a TEM grid. In many cases, the number of holes with good sample quality and particle distribution is limited. Therefore, it is important to obtain as much high-quality data as possible given the limited amount of good-quality sample. Therefore, there is a need for a highly efficient, effective, and high-throughput data collection scheme using relatively low-cost detectors.

[0011] FIG. 2 shows an exemplary scheme for acquiring SPA cryo-EM data from a single 2 μm diameter hole 201 in a TEM grid. The hole is imaged using a circular beam (forming a circular illumination zone) at five beam locations shown as illumination regions 202-206. A detector with a square detection array is used for data collection. Within each of the circular illumination regions 202-206, radiation emitted from a respective square detection zone (207-211) is detected by the detector. The illumination regions are arranged such that the detection zones are in areas of the sample that are illuminated only once to avoid collecting data from radiation damaged samples (i.e. overlapping regions of the illumination area). As a result, a relatively large area of ​​the hole 201 is not covered by any of the detection zones and data from samples (e.g. particles) located in these regions is not collected. In other words, in this example, data collection efficiency and effectiveness is low.

[0012] To address the above problems, a charged particle beam is directed towards a sample positioned in a sample plane, so that an illumination zone is formed in the sample plane. The illumination zone is an area illuminated by the charged particle beam and extends along a first illumination axis and a second illumination axis in the sample plane. The illumination zone may be of any shape, such as rectangular, square, circular or elliptical. The illumination zone is scanned / moved continuously or discretely in a substantially first direction in the sample plane.

[0013] In response to the illumination, the detector receives radiation (such as charged particles, X-rays, or electromagnetic radiation) from a detection zone in the sample plane. The detection zone extends along a first detection axis and a second detection axis. The detection zone may be located within the illumination zone. The size, shape, and relative position of the detection zone with respect to the illumination zone are determined and can be adjusted by the configuration of the system. The radiation from the detection zone is detected / received by a plurality of detection pixels of the detector. The plurality of detection pixels are arranged along a first detector axis and a second detector axis of the detector to detect the spatially resolved radiation. The first detector axis corresponds to or is aligned with the first detection axis, and the second detector axis corresponds to or is aligned with the second detection axis. In other words, radiation along the first detector axis is resolved along the first detector axis, and radiation along the second detection axis is resolved along the second detector axis. The first detection axis is oriented at an angle of less than 45 degrees from the first direction. If the first sensing axis is non-zero degrees from the first direction, then the sensing zone is tilted from (or does not coincide with) the first direction.

[0014] The first number of the plurality of detector pixels arranged along the first detector axis is less than the second number of the plurality of detector pixels arranged along the second detector axis. In one embodiment, the number of detector pixels arranged along the first detector axis may be one. That is, the plurality of detector pixels are arranged in a 1D array. In another embodiment, the width of the detection zone along the first detector axis is less than the length of the detection zone along the second detector axis. In yet another embodiment, the width of the detector array formed by the plurality of detector pixels along the first detector axis is less than the length of the detector array along the second detector axis. Thus, the number of detector pixels (or active detector pixels) used to detect radiation may be reduced. In some embodiments, all detector pixels of the detector are used to detect radiation. In some embodiments, a subset of the detector pixels of the detector are used to detect radiation. In other embodiments, multiple different subsets of the detector pixels of the detector are used during a single scan.

[0015] In some embodiments, the illumination zone may be continuously scanned or moved in the sample plane substantially along a first illumination axis in a first direction, and radiation from the sample is acquired by a detector. In other embodiments, the sample is scanned substantially in a first direction at multiple beam locations using a charged particle beam. At each beam location, the illumination zone is illuminated with the charged particle beam, and radiation from a detection zone within the illumination zone is detected by a detector. By "substantially" scanning along the first illumination axis (or in the first direction), the actual scan path may deviate slightly from the first illumination axis (or in the first direction), but may substantially follow the first illumination axis (or in the first direction). For example, the actual beam position at a given time during the scan may deviate slightly from the first direction, but a linear interpolation of the actual beam position is within 10 degrees of the first illumination axis.

[0016] In one embodiment, the charged particle beam is directed at the sample to form an illumination zone in the sample plane, and a first radiation from the detection zone of the sample is detected using a plurality of detection pixels of the detector arranged along a first detector axis and a second detector axis. The first number of the plurality of detection pixels arranged along the first detector axis is less than the second number of the plurality of detection pixels arranged along the second detector axis. The illumination zone and the detection zone are moved in a first direction in the sample plane, and a second radiation from the detection zone is detected using the plurality of detection pixels. The first detection axis of the detection zone corresponds to the first detector axis, and the second detection axis of the detection zone corresponds to the second detector axis, and the angle between the first direction and the first detection axis is less than 45 degrees. The sample image is reconstructed based on the first radiation and the second radiation. In some embodiments, after detecting the second radiation, the illumination zone and the detection zone are moved in a second direction, and a third radiation from the detection zone is detected. The second direction may be determined based on sample / system drift. In some embodiments, the charged particle beam may be adjusted based on sample / system drift while moving the irradiation zone. The second direction may be less than 45 degrees from the first direction. An image of the sample is further reconstructed based on the third detected radiation.

[0017] The angle between the first detection axis and the first direction may be less than 45 degrees, less than 30 degrees, or less than 10 degrees. In some embodiments, the first detection axis is substantially aligned or parallel to the first direction.

[0018] The charged particle beam or the illumination zone can be scanned / moved substantially along a first illumination axis by adjusting one or more beam deflection coils that direct the charged particle beam towards the sample and / or a beam limiting aperture in the beam path of the charged particle beam. While scanning the illumination area (or the charged particle beam) in the first direction, the relative position of the detection area with respect to the illumination area does not change, i.e. the detection zone is scanned in the sample plane at the same speed as the illumination zone.

[0019] In some embodiments, the scanning path of the illumination zone may deviate slightly from the first direction due to factors including, for example, the illumination of the electron beam, the orientation of the detector, and the sample / hole structure. The sample image may be reconstructed based on the scanning direction and scanning speed of the illumination zone. In some embodiments, drift or deviation from the scanning path may be monitored during the scan, and based on the drift, the position of the illumination zone in the sample plane and / or the first direction (e.g., the scanning direction) and / or the optical components for generating the charged particle beam may be adjusted. The drift may be detected, for example, based on the reconstructed image of a portion of the sample.

[0020] The sample image may be reconstructed based on time information of the received radiation. For example, the received radiation may be spatially resolved along the first illumination axis based on the time the radiation is detected by the detector. In some embodiments, the particles of radiation are sampled in time by event-based detection. The sample image may be reconstructed further based on information about the movement of the charged particle beam. For example, the sample image may be reconstructed based on the direction of the illumination zone and the scanning speed. The sample image may further be reconstructed based on information of the detected radiation. For example, the sample image may further be reconstructed based on the speed of the detected electrons. In some embodiments, the sample image is reconstructed based on the scanning speed, the scanning direction, and the time of the detected event.

[0021] In some embodiments, one or more inactive pixels may be positioned between a plurality of detection pixels for detecting radiation from the detection zone. The inactive pixels may be defective pixels in the 2D detector array. In some examples, one or more empty spaces may be positioned between a plurality of detection pixels. Because the illumination zone is scanned over the sample and the image is reconstructed based on time information of radiation received from different parts of the pixelated array, the methods disclosed herein are insensitive to defective pixels in the detector array.

[0022] In this way, the number of detector pixels for detecting radiation can be reduced without sacrificing the quality performance of the detector. Compared to DEDs with a wide field of view, a detector with a smaller number of detector pixels can be used here, and a cheaper detector can be acquired with a smaller number of detector pixels. In some embodiments, the number of detector pixels along the second detector axis may be 100-200 times the number of detector pixels along the first detector axis. For example, the number of detector pixels may be 2 along the first detector axis and 128-1024 along the second detector axis. Furthermore, the spatial resolution of the detector along the first detector axis may be much lower than the second detector axis. By reducing the detector requirements, the cost of the detector can be further reduced.

[0023] In some embodiments, the first detector axis and the second detector axis are mutually orthogonal. The first detection axis and the second detection axis are mutually orthogonal. The illumination zone may be formed by a beam limiting aperture positioned in the beam path of the charged particle beam. One example of forming an illumination zone is disclosed in U.S. Pat. No. 1,143,0633 (B2), the entirety of which is incorporated herein by reference for all purposes.

[0024] The plurality of detection pixels of the detector may be a subset of the detection pixels of the detector, for example a rectangular array of detection pixels in a circular, rectangular, or square pixelated detector may be active for detecting radiation.

[0025] The detection zone is part or all of the illumination zone, and radiation from the detection zone is acquired by a detector and used to form an image of the sample. In some embodiments, the detection zone is smaller and is within the illumination zone. By having an illumination zone that is larger than the detection zone, the illumination zone can extend to overlap conductive material surrounding the sample region, avoiding charge build-up in the sample region.

[0026] The detection zone may be bounded by first and second detection edges parallel to the second detection axis and third and fourth detection edges parallel to the first detection axis. The illumination zone may be bounded by first and second illuminated edges and third and fourth illuminated edges oriented along a first direction. The first and first illuminated edges are disposed toward the first direction relative to the second and second illuminated edges.

[0027] In some examples, one or more of the detection edge and the illumination edge may be straight. In some examples, one or more of the detection edge and the illumination edge may be curved. The location of the detection zone relative to the illumination zone may be adjusted by adjusting one or more image deflection coils positioned between the sample and / or by adjusting the position and orientation of the detector. In some embodiments, one or both of the illumination zone and the detection zone are rectangular.

[0028] During at least a portion of the scanning and data collection process, a portion of the illumination zone may overlap with a conductive material surrounding the sample area. In this way, charge may be removed from the sample during scanning. In one embodiment, the sample is vitrified within the sample area surrounded by a support material (such as carbon). The support material surrounding the sample area may be conductive. The sample area may be of any shape. For example, the sample area may be rectangular, square, or circular. In one embodiment, the sample area may be a hole in a TEM grid.

[0029] The detection zone may be offset relative to the illumination zone. In other words, at least one of the central axes of the detection zone does not coincide with the central axis of the illumination zone. The detection zone may be offset relative to the illumination zone toward the scanning direction (e.g., the first direction). In some embodiments, at least one of the central axes of the detection zone parallel and perpendicular to the first direction does not coincide with the central axes of the illumination zone parallel and perpendicular to the first direction, respectively. In one embodiment, the detection zone is offset in the scanning direction relative to the illumination zone. By offsetting the detection zone toward the scanning direction, the illumination zone may extend and overlap the material opposite to the scanning direction, thus reducing potential radiation damage to the unscanned sample area. In one embodiment, a first distance between the first detection edge and the first illumination edge is smaller than a second distance between the second detection edge and the second illumination edge. The first distance may be non-zero and may be determined based on the speed of the scan along the first axis and the estimated flux of radiation received. By keeping the first distance non-zero, initial radiation generated from a particular illumination zone during scanning is not collected. This is because these initial rays can introduce beam-induced particle motion and degrade the quality of the received signal. One of the third detection edge and the fourth detection edge can substantially overlap one of the third illumination edge and the fourth illumination edge.

[0030] In one embodiment, after scanning along a first direction, data from a first row of the sample area is collected. Then, the illumination zone is shifted to collect data from a second row of the sample area. The first and second rows in the sample area are parallel and may or may not overlap. The collected data may be selected during post-processing for image reconstruction. To collect data from the second column, the illumination zone may be shifted in the second direction by shifting the charged particle beam via adjusting the beam deflection coils and / or by moving / shifting the sample by adjusting the sample stage. The illumination zone may also be shifted by moving an aperture positioned in the charged particle beam path and upstream of the sample plane. While scanning the illumination zone, post-beam corrections are applied to adjust the relative position between the illumination zone and the detection zone.

[0031] In this manner, data from a large portion of the sample area can be collected, thus achieving efficient, effective and high throughput data collection.

[0032] Referring to FIG. 1, a highly simplified TEM system 101 is shown. An electron beam 103 generated by an electron source 116 is limited by an aperture 104 before being deflected by a beam deflection coil 105. The beam deflection coil may shift or move the area in the sample plane 110 (XY plane) where the electron beam is irradiated (i.e., the irradiation zone). The deflected beam passes through an upper objective pole piece 106 into an upper opening 107 of an optional cryobox 108. A sample (not shown) positioned at one end of a sample holder 109 is irradiated by the electron beam entering through the upper opening 107. The beam profile at the sample plane, which forms the irradiation zone, is determined in part by the beam limiting aperture 104. The position of the sample can be adjusted by operating one or more actuators of the stage 102. For example, the stage 102 may be operated to move the sample in the sample plane via the sample holder. Radiation 120 from the sample exits the cryobox 108 through the bottom opening 112 of the cryobox. The radiation 120 passes through the lower objective pole piece 113, is deflected by image deflection coils 114 in descanned mode, and then reaches the detector 115. The detector 115 may be a pixelated detector having multiple detector elements for receiving the radiation. FIG. 3C shows an example of an arrangement of multiple detector elements. In one example, the detector 115 is a pixelated detector with particle counting and tracking capabilities. For example, the detector is a Timepix-based detector. In another example, the detector is an active pixel CMOS direct electron detection camera.

[0033] Signals from the detector 115 are sent along control lines (buses) to a controller 117 for processing and forming a sample image. The sample image may be displayed on a display unit (not shown). Such processing may include operations such as combining, integrating, subtracting, false coloring, edge enhancement, and other processing known to those skilled in the art. The controller includes a non-transitory memory 119 for storing computer readable instructions and a processor 118. The methods disclosed herein may be implemented by executing computer readable instructions stored in the non-transitory memory 119 in the processor 118. For example, the controller may control the microscope to direct an electron beam at the sample, collect data, and process the collected data. The controller may adjust the electron beam energy, position, illumination area, and intensity by adjusting one or more lenses and / or electron sources. The controller may adjust the sample position via the sample holder and stage. The controller may adjust the location of the illumination zone via image deflection coils. The controller may adjust the sample area imaged (e.g., the location of the detection zone) via image deflection coils.

[0034] Although a TEM system is shown here as an example of a charged particle microscope, the methods and system configurations disclosed herein can be applied to other types of charged particle microscopes.

[0035] Figures 3A and 3B illustrate two exemplary arrangements of illumination zones and detection zones. Figure 3C illustrates one exemplary arrangement of detector elements of a detector that can be used to detect radiation from the detection zones shown in Figures 3A and 3B. In these examples, both the illumination zones and the detection zones are rectangular.

[0036] In Figures 3A and 3B, the illumination zone 320 is the area in the sample plane where the charged particle beam is illuminated. Coordinate system 380 shows the coordinates of the illumination zone, and coordinate system 381 shows the coordinates of the detection zone, where the illumination coordinates and the detection coordinates are identical. The Y axis is the first illumination axis and the X axis is the second illumination axis. The illumination zone is scanned in a direction 350 along the first illumination axis (Y axis of 380, 381) in the sample plane. The detection zone 310 is within and smaller than the illumination zone 320. The first detection axis is the same as the first illumination axis and the second detection axis is the same as the second illumination axis. The width 304 (along the scanning direction 350) of the detection zone is smaller than the width 302 of the illumination zone. The length 301 of the illumination zone may be larger or the same as the length 303 of the detection zone. The illumination zone is bounded by a first illuminated edge 309, a second illuminated edge 313, a third illuminated edge 315, and a fourth illuminated edge 314. The illumination zone has a width 302 and a length 301. The detection zone is bounded by a first detection edge 307, a second detection edge 316, a third detection edge 318, and a fourth detection edge 317. The detection zone has a width 304 and a length 303.

[0037] In both Figures 3A and 3B, the detection zone 310 is offset from the illumination zone 320 in the scan direction 350. The central axis 308 of the detection zone 310 does not overlap with the central axis 305 of the illumination zone 320. The distance between the first illumination edge 309 and the first detection edge 307 is smaller than the distance between the second illumination edge 316 and the second detection edge 313. In this way, the illumination zone 320 may extend in a direction opposite to the scan direction 350 (opposite the direction of the Y-axis) to cover a portion of the material surrounding the sample area and facilitate charge removal. Furthermore, the distance between the first illumination edge 309 and the first detection edge 307 may be greater than zero such that initial radiation generated in response to illumination is not detected by the detector.

[0038] Here, the central axis 321 of the detection zone 310 does not overlap with the central axis 306 of the illumination zone 320. The detection zone 310 may be offset toward different directions along the X-axis as shown in Figures 3A and 3B. In other words, the distance between the third illumination edge 315 and the third detection edge 318 may be smaller (Figure 3A) or larger (Figure 3B) than the distance between the fourth illumination edge 314 and the fourth detection edge 317. The direction in which the detection zone is offset from the illumination zone may determine which portion of the sample area is scanned, such that the illumination zone may extend to cover the material surrounding the sample area as well as the sample area already scanned. In some embodiments, the third illumination edge 315 may overlap with the third detection edge 318 and / or the fourth illumination edge 314 may overlap with the fourth detection edge 317.

[0039] FIG. 3C illustrates an exemplary arrangement of multiple detector elements (or pixels) of a pixelated detector for receiving / acquiring radiation from the detection zone of FIG. 3A and FIG. 3B. Coordinate system 382 is the coordinate system of the detector. The detector elements are arranged in a 2D array extending along a first detector axis (Y-axis of 382) and a second detector axis (X-axis of 382). The first detector axis corresponds to the first detector axis (Y-axis of 381), and the second detector axis corresponds to the second detector axis (X-axis of 381). The detector elements (e.g., 330-337) arranged along the second detector axis can detect spatially resolved radiation along the second detector axis. The detector elements (e.g., 330, 341, 342, and 343) arranged along the first detector axis can detect spatially resolved radiation along the first detector axis. The number of detector elements along the first detector axis is less than the number of detector elements along the second detector axis. The shape of each detector element may be square or rectangular. In some embodiments, the detector elements may be other shapes. The spatial resolution of each detector element may be higher along the second detector axis compared to the first detector axis. In one embodiment, the first detector axis is aligned with the scan direction 350. In some embodiments, the detector elements shown in FIG. 3C may be a subset of the detector elements of the detector.

[0040] In some embodiments, the detector elements are arranged in a 1D array, in other words, the number of detector elements along the first detection axis is one, in some embodiments, the illumination zone may be other shapes, such as circular, square, or elliptical.

[0041] In some embodiments, the scanning direction may not coincide with the detection axis or the illumination axis. For example, the illumination zone 320 may be scanned along a direction having a non-zero angle from the direction 350. In some embodiments, the detection axis may have an angle of less than 45 degrees from the scanning direction.

[0042] 3D illustrates another exemplary arrangement of the illumination zone 351 and the detection zone 352. The detection zone is rectangular and is defined by a first detection axis (the Y axis of the detection coordinate system 381) and a second detection axis (the X axis of the detection coordinate system 381). The detection coordinate system 381 does not coincide with the illumination coordinate system 380. The scanning direction 350 does not coincide with either the first detection axis (the Y axis of the detection coordinate system 381) or the first illumination axis (the Y axis of the illumination coordinate system 381). The first detection axis (the Y axis of the detection coordinate system 381) has an angle 355 from the scanning direction 350. The angle 355 is less than 45 degrees.

[0043] 4A-4H illustrate one exemplary scanning scheme for collecting data from a circular sample area. The sample area 401 here is a 2 μm diameter hole in a TEM grid. A sample (e.g., containing multiple particles) can be vitrified in the hole and imaged using the TEM system of FIG. 1. As shown sequentially in FIGS. 4A-4E, the left side (e.g., left side relative to the central axis 409) of the sample area 401 is scanned with the illumination zone 402 and data is collected from the corresponding detection zone 403. Then, as shown sequentially in FIGS. 4F-4H, the right side (e.g., right side relative to the central axis 409) of the sample area is scanned with the illumination zone 410 and data is collected from the corresponding detection zone 411. The relative arrangement of the illumination zones and detection zones can be the same as shown in FIGS. 3A-3B.

[0044] In Figure 4A, at the very first beam location, the illumination zone 402 is positioned at the top left corner of the sample area 401. At the next beam location shown in Figure 4B, the beam shift moves the illumination zone in the scan direction along the first illumination axis (Y-axis). As the illumination zone scans further along the first illumination axis, sample data is collected from a first row 406 of the sample area. A sample image generated based on the data collected from the first row 406 is shown in Figure 4C. At each beam location for scanning the first row, the illumination zone overlaps with an area outside the sample area 401 to avoid charge accumulation.

[0045] In FIG. 4D, the illumination zone 402 is shifted along the second illumination axis (X-axis) toward the right side of the first row. In addition, the illumination zone 402 is shifted back along the first illumination axis to start data collection from the second row 407 of sample areas. The shift of the illumination zone from FIG. 4C to FIG. 4D may be performed by beam shifting via the beam deflection coils and / or sample shifting via the sample holder. Additionally or alternatively, the shift can be achieved by adjusting the beam limiting aperture. The illumination zone 402 scans along the scanning direction (Y-axis direction) and ends scanning the second row 407 in FIG. 4E. In some embodiments, the second row 407 may be acquired by scanning the illumination zone in the opposite direction to the Y-axis direction. In this case, the detection zone is offset in the scanning direction (opposite to the Y-axis direction) with respect to the illumination zone.

[0046] The relative position of the detection zone 403 with respect to the illumination zone 402 is shown in Figure 3A, where the detection zone is offset towards the unscanned sample area (right side), so that the non-overlapping area between the illumination zone and the detection zone is mostly within the sample area already scanned, thereby minimizing radiation damage to the unscanned sample area.

[0047] In FIG. 4F, compared to the location of the illumination zone 402, the illumination zone 410 is shifted along the second illumination axis (X-axis) toward the right side of the first column. In addition, the illumination zone 410 is shifted back along the first illumination axis to start data collection from the third column 412 of the sample area. At the same time, the relative position of the detection zone 411 from the illumination zone 410 is different from that shown in FIG. 4A-FIG. 4E. In FIG. 4E, the detection zone is offset toward the left side of the illumination zone, as shown in FIG. 3B. Thus, the illumination zone 410 can overlap with the area surrounding the sample area 401, and can also reduce radiation damage to the unscanned sample area. The illumination zone 410 is scanned in the scanning direction along the first illumination axis to complete data collection of the third column 412, as shown in FIG. 4G. Then, the illumination zone is shifted along the second illumination axis and scanned along the first illumination axis to complete data collection of the fourth column 413, as shown in FIG. 4H.

[0048] 4A-4H show the beam locations as discrete locations, scanning along the first illumination direction may be continuous, i.e., the illumination zone is continuously moved or scanned along the first illumination axis at a non-zero scan speed.

[0049] Figure 5 is an integrated diagram of the locations of the illumination and detection zones during scanning of a sample area 510. Similar to Figures 4A-H, two relative positions between the illumination and detection zones are used to scan different sides of the sample area. Unlike Figures 4A-H, the sample area is covered by six columns (i.e. scanned six times along the first illumination direction).

[0050] Figure 6 is a close-up view of the upper left region of Figure 5. The first detection edge 602 does not overlap with the first illumination edge 601. Therefore, the first few electrons generated in the sample area between the first detection edge 602 and the first illumination edge 601 are not detected. The distance between the two edges may be positioned based on the speed of the scan along the first axis and the estimated flux of received radiation.

[0051] FIG. 7 illustrates a method 700 for imaging a sample using a charged particle microscope, such as the TEM system shown in FIG.

[0052] At 702, a sample is loaded into a charged particle microscope and parameters for imaging the sample are set. In one embodiment, the sample is a vitrified sample positioned on a TEM grid. The sample may be loaded into the microscope using a sample holder. Loading the sample may include positioning the sample in the sample plane and in the beam path. In some embodiments, one or more sample images may be acquired to assist in positioning the sample. Parameters for imaging the sample may include one or more of a beam current, a scan path, a scan speed, a size of the illumination zone and the detection zone, and a position of the detection zone relative to the illumination zone. The parameters may be determined based on the type of sample. For example, based on a user input of the sample type, the scan speed may be determined based on the dose limit of the sample type. The flux of radiation received from the sample may be estimated based on the sample type and the sample distribution.

[0053] In one embodiment, the size and shape of the illumination zone may be selected based on one or more of a region of interest (ROI) to be imaged on the sample, dose limits, and scan speed. The ROI may be any shape, such as circular or rectangular. Additionally, a position of the detection zone relative to the illumination zone may be determined. In some embodiments, the size of one or both of the illumination zone and the detection zone may change during the scan. In some embodiments, the relative position of the detection zone with respect to the illumination zone remains the same throughout the scan. The position of the detection zone with respect to the illumination zone may be determined based on one or more of an estimated flux, a scan speed, a sample type, and a scan path. For example, the distance between the first detection edge and the first illumination edge may increase with the speed of the scan and decrease with an increase in the estimated flux. As another example, the offset of the detection zone with respect to the illumination zone may depend on the scan path as shown in FIGS. 4A-4H. In yet another embodiment, the scan direction may change during the scan. For example, the scan may have a serpentine scan path. In some embodiments, one or more test scans may be performed, for example, on a test sample to adjust one or more of the imaging parameters prior to imaging the sample of interest.

[0054] In 703, the charged particle beam is directed towards the sample via beam deflection coils to form an illumination zone in the sample plane. Step 703 may include selecting a location of the detection zone relative to the illumination zone from 702 and determining corresponding parameters for the beam deflection coils and image deflection coils.

[0055] In 704, the illumination zone is scanned in a first direction along a first illumination axis in the sample plane according to the parameters set in 702, and radiation from the detection zone is collected by the detector. The scan may be a stepwise scan or a continuous scan. In some embodiments, the sample image may be displayed and updated while collecting data. In some examples, drift may be estimated during the scan. Drift may include system drift and sample drift. To correct for drift, the scan path or the location of the illumination zone may be adjusted based on the estimated drift.

[0056] At 706, method 700 checks whether data acquisition along the first illumination axis is complete (i.e., data collection along one row is complete). If the answer is yes, method 700 moves to 708. If not, scanning continues in the first direction.

[0057] At 708, the method 700 checks whether data collection from the ROI is complete. If the answer is yes, at 710, one or more sample images are reconstructed based on the collected data. If data collection from the ROI is not complete, the illumination zone is repositioned on the sample plane at 712 to continue data collection of another area within the ROI. For example, at 712, the illumination zone may be shifted along a second illumination axis to collect data from another column of the ROI. The position of the detection zone relative to the illumination zone may be changed at step 703 to collect radiation corresponding to the repositioned illumination zone.

[0058] The technical effect of scanning the illumination zone to acquire radiation from the detection zone and detecting radiation through a detector with a first number of multiple detection pixels arranged along a first detector axis that is less than a second number of multiple detection pixels arranged along a second detector axis is that the cost of the detector can be reduced while obtaining a sample image with high spatial resolution. By using a special detection zone shape (formed by an aperture), the method allows for high efficiency sample area sampling and reduced sample damage. Furthermore, by scanning the illumination zone, drift correction can be applied via real-time feedback. The method can scan a larger FOV compared to standard methods. The technical effect of having a detection zone that is within the illumination zone and smaller than the illumination zone is that the illumination zone can cover material surrounding the sample area for charge removal. The technical effect of constructing a sample image based on time information of received radiation and the scanning speed is that the spatial resolution requirement of the detector along the first detector axis is lower. Furthermore, the beam can be scanned continuously along the scanning direction, thus reducing the overall data acquisition duration. In addition, the method is insensitive to the number of defective pixels in the detector array and to imperfections in the illumination.

Claims

1. 1. A method for imaging a sample, comprising: directing a charged particle beam towards the sample to form an irradiation zone in a sample plane; scanning the illumination zone in substantially a first direction within the sample plane and detecting radiation from the detection zone using a plurality of detection pixels of a detector, a first detector axis of the detector corresponding to a first detection axis of the detection zone and a second detector axis of the detector corresponding to a second detection axis of the detection zone, a first number of the plurality of detection pixels disposed along the first detector axis being less than a second number of the plurality of detection pixels disposed along the second detector axis, and the first detection axis being oriented at an angle of less than 45 degrees from the first direction; and reconstructing an image of the sample based on the radiation detected during the scan.

2. The method of claim 1 , wherein scanning the illumination zone substantially in the first direction comprises shifting the charged particle beam substantially in the first direction relative to the sample.

3. The method of claim 1 , wherein the size, shape, and relative positions of the illumination zone and the detection zone remain the same while scanning the illumination zone substantially in the first direction.

4. The method of claim 1 , wherein the detection zone is within the illumination zone and is offset relative to the illumination zone in the first direction.

5. 5. The method of claim 4, wherein the detection zone is bounded by first and second detection edges parallel to the second detection axis, the illumination zone is bounded by first and second illuminated edges perpendicular to the first direction, the first detection edge and the first illuminated edge are disposed toward the first direction relative to the second detection edge and the second illuminated edge, and a first distance between the first detection edge and the first illuminated edge is less than a second distance between the second detection edge and the second illuminated edge.

6. 6. The method of claim 5, wherein the first distance between the first detection edge and the first illumination edge is determined based on a speed of the scanning of the illumination zone in the first direction and an estimated flux of the detected radiation.

7. 5. The method of claim 4, wherein the detection zone is bounded by third and fourth detection edges parallel to the first detection axis, and the illumination zone is bounded by third and fourth illuminated edges oriented along the first direction, one of the third and fourth detection edges substantially overlapping one of the third and fourth illuminated edges.

8. 8. The method of claim 1, further comprising: after scanning the sample over a first distance substantially along the first direction, shifting the illumination zone over a second distance in a second, different direction; and scanning the shifted illumination zone substantially in the first direction and detecting radiation from the detection zone.

9. 2. The method of claim 1, wherein scanning an illumination zone in the first direction within the sample plane comprises continuously moving the illumination zone in the first direction, and reconstructing a sample image based on the radiation detected during the scan comprises reconstructing a location of the detected radiation along the first detection axis based on time information of the detected radiation and a scanning speed of the illumination zone in the first direction.

10. 1. A method for imaging a sample, comprising: directing a charged particle beam at the sample to form an illumination zone in a sample plane and detecting a first radiation from the detection zone of the sample using a plurality of detection pixels of a detector arranged along a first detector axis and a second detector axis, a first number of the plurality of detection pixels arranged along the first detector axis being less than a second number of the plurality of detection pixels arranged along the second detector axis; moving the illumination zone and the detection zone in a first direction within the sample plane and detecting a second radiation from the detection zone using the plurality of detection pixels, wherein a first detection axis of the detection zone corresponds to the first detector axis, a second detection axis of the detection zone corresponds to the second detector axis, and an angle between the first direction and the first detection axis is less than 45 degrees; and reconstructing an image of the sample based on the first ray and the second ray.

11. 11. The method of claim 10, further comprising: after detecting the second radiation, moving the illumination zone and the detection zone in a second direction and detecting a third radiation from the detection zone; and reconstructing the sample image further based on the third radiation.

12. 1. A charged particle microscope, comprising: a sample holder for positioning the sample in a sample plane; a charged particle column for directing a charged particle beam towards the sample plane to form an irradiation zone in the sample plane; a pixelated detector for detecting radiation produced from a detection zone of the sample in response to irradiating the sample with the charged particle beam, a first detector axis of the detector corresponding to a first detection axis of the detection zone and a second detector axis of the detector corresponding to a second detection axis of the detection zone; a controller, the controller including a non-transitory memory for storing computer readable instructions, and a processor, the computer readable instructions being executed in the processor to cause the charged particle microscope to: scanning the illumination zone in substantially a first direction in the sample plane through the charged particle column; detecting radiation from the detection zone through a plurality of pixels of the detector, wherein a first number of the plurality of detection pixels arranged along the first detector axis is less than a second number of the plurality of detection pixels arranged along the second detector axis, and the angle of the first direction from the first detection axis is less than 45 degrees; a charged particle microscope configured to reconstruct an image of the sample based on the radiation detected during the scan.

13. 13. The charged particle microscope of claim 12, wherein the charged particle column directs a charged particle beam towards the sample plane via one or more beam deflection coils and a beam limiting aperture in the charged particle column, and the charged particle column scans the irradiation zone by adjusting the beam deflection coils and / or the beam limiting aperture.

14. 14. The charged particle microscope of claim 13, further comprising one or more image deflection coils for directing radiation from the detection zone towards the detector, the relative position of the detection zone within the illumination zone being adjusted by the one or more image deflection coils.

15. The charged particle microscope of claim 12 , wherein the charged particle microscope comprises a transmission electron microscope system.

16. 13. The charged particle microscope of claim 12, wherein the detector includes a first number of pixels, and detecting radiation from the detection zone includes detecting the radiation from the detection zone using a second, fewer number of the pixels of the detector.

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