Method and system for imaging sample

Abstract

The present disclosure relates to methods and systems for imaging a sample. The sample is imaged by directing the charged particle beam toward the sample and forming an irradiation region. The charged particle beam is scanned such that the irradiation region is scanned in a first direction in the sample plane and radiation from the detection region is detected by a detector. A first number of detection pixels arranged along a first detector axis corresponding to the first detection axis is less than a second number of detection pixels arranged along a second detector axis corresponding to the second detection axis.

Description

Technical Field

[0001] The present specification 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 toward the sample and forming an irradiation region in a sample plane; scanning the irradiation region in the sample plane substantially in a first direction, and detecting radiation from the detection region with a plurality of detection pixels of a detector, wherein a first detector axis of the detector corresponds to a first detection axis of the detection region, and a second detector axis of the detector corresponds to a second detection axis of the detection region, wherein a first number of the plurality of detection pixels arranged along the first detector axis is lower than a second number of the plurality of detection pixels arranged along the second detector axis, and wherein the first detection axis is oriented at an angle less than 45 degrees to the first direction; and reconstructing an image of the sample based on radiation detected during the scan.

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

[0004] In 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 and forming an irradiation region in the sample plane; a pixelated detector for detecting radiation generated from a detection region of the sample in response to irradiating the sample with the charged particle beam, wherein a first detector axis of the detector corresponds to a first detection axis of the detection region, and a second detector axis of the detector corresponds to a second detection axis of the detection region; and a controller including a processor and a non-volatile memory for storing computer-readable instructions, wherein by executing these computer-readable instructions in the processor, the charged particle microscope is configured to: scan the irradiation region in a substantially first direction in the sample plane via the charged particle column; detect radiation from the detection region via a plurality of pixels of the detector, wherein a first number of the plurality of detection pixels arranged along the first detector axis is lower than a second number of the plurality of detection pixels arranged along the second detector axis, and wherein the first direction is at an angle of less than 45 degrees to the first detection axis; and reconstruct an image of the sample based on radiation detected during the scan.

[0005] It should be understood that the above summary is provided to introduce some concepts otherwise described in the detailed description in a simplified form. It is not intended to identify key or essential features of the claimed subject matter, the scope of which is solely defined by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to implementations that address any disadvantages mentioned above or in any part of this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 An example of a charged particle microscope is shown.

[0007] Figure 2 An example of collecting data from a circular sample area is shown.

[0008] FIG. 3A to FIG. 3B Example irradiation areas and corresponding detection areas are shown.

[0009] Figure 3C An example arrangement of detection pixels of a detector is shown.

[0010] Figure 3D Another example irradiation region and corresponding detection region is shown.

[0011] 4A to 4H An example data collection scenario is shown.

[0012] Figure 5Another example of collecting data from a circular sample area is shown.

[0013] Figure 6 Shows Figure 5 part.

[0014] Figure 7 An example method for imaging a sample is shown.

[0015] Like reference numerals refer to corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION

[0016] The following description relates to systems and methods for imaging samples using charged particle microscopy. Charged particle microscopy can be used to analyze sample features and structures at high resolution. For radiation sensitive samples, high quality sample imaging with low doses of radiation can be challenging. However, detectors with high sensitivity and low noise levels can be expensive.

[0017] As an example, a transmission electron microscope (TEM) is a charged particle microscope that can provide high-resolution details of biological structures. One application of TEM is for single particle analysis (SPA), where the structure of particles such as proteins or viruses can be reconstructed. Because samples used for SPA are typically highly radiation sensitive, a lower dose of radiation is required during the imaging period. Direct electron detectors (DEDs) are the leading 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), are out of reach for potential users due to their high cost.

[0018] In addition, the process for sample preparation can be very complex and time consuming. For example, in SPA, the sample (e.g., in the form of particles) is vitrified and imaged in the wells of a TEM grid. Typically, the number of wells with good sample quality and particle distribution is limited. Therefore, given the limited amount of high-quality samples, it is critical to obtain as much high-quality data as possible. Therefore, a high-efficiency, high-effectiveness, and high-throughput data collection scheme using relatively low-cost detectors is required.

[0019] Figure 2An example scheme for acquiring SPA cryo-EM data from a single 2 μm diameter hole 201 of a TEM grid is shown. The hole is imaged using a circular beam (which forms a circular irradiation area) at five beam positions shown as irradiation areas 202 to 206. Data collection is performed using a detector with a square detection array. Within each of the circular irradiation areas 202 to 206, radiation originating from the corresponding square detection area (207 to 211) is detected by the detector. The irradiation areas are arranged so that the detection areas are in a sample area that is irradiated only once, so as to avoid collecting data from radiation-damaged samples (i.e., overlapping areas of the irradiation areas). As a result, relatively large areas of the hole 201 are not covered by any of the detection areas, and data from samples (e.g., particles) located in these areas are not collected. In other words, in this example, data collection efficiency and effectiveness are low.

[0020] In order to solve the above problems, a charged particle beam is directed toward a sample positioned in a sample plane so that an irradiation area is formed in the sample plane. The irradiation area is an area irradiated by the charged particle beam and extends along a first irradiation axis and a second irradiation axis in the sample plane. The irradiation area can be any shape, such as a rectangle, a square, a circle or an ellipse. The irradiation area is scanned / moved continuously or discretely in a first direction in the sample plane.

[0021] In response to the irradiation, the detector receives radiation (such as charged particles, X-rays, or electromagnetic radiation) from a detection area in the sample plane. The detection area extends along a first detection axis and a second detection axis. The detection area can be located within the irradiation area. The size, shape, and relative position of the detection area relative to the irradiation area can be determined and can be adjusted by the configuration of the system. Radiation from the detection area 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 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 detection 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 less than 45 degrees to the first direction. When the first detection axis is non-zero degrees to the first direction, the detection area is tilted from the first direction (or not aligned with the first direction).

[0022] A first number of multiple detection pixels arranged along the first detector axis is less than a second number of multiple detection pixels arranged along the second detector axis. In one example, the detection pixel arranged along the first detector axis can be one. That is, the multiple detection pixels are arranged in a 1D array. In other examples, the width of the detection area along the first detection axis is shorter than the length of the detection area along the second detection axis. In yet another example, the width of the detector array formed by the multiple detection pixels along the first detector axis is shorter than the length of the detector array along the second detector axis. In this way, the number of detection pixels (or active detection pixels) used to detect radiation can be reduced. In some examples, all detection pixels of the detector are used to detect radiation. In some examples, a subset of the detection pixels of the detector are used to detect radiation. In other examples, multiple different subsets of the detection pixels of the detector are used during a single scan.

[0023] In some examples, the irradiation area can be continuously scanned or moved in a first direction substantially along a first irradiation axis in a sample plane, and radiation from the sample is acquired by a detector. In other examples, a charged particle beam is used to scan the sample at multiple beam positions substantially in a first direction. At each beam position, the irradiation area is irradiated with a charged particle beam, and radiation from a detection area within the irradiation area is detected by a detector. By scanning "substantially" along the first irradiation axis (or in the first direction), the actual scanning path may deviate slightly from the first irradiation axis (or in the first direction) but substantially follow the first irradiation axis (or in the first direction). For example, the actual beam position at a specific point in time during the scan may be slightly shifted from the first direction, but the linear interpolation of the actual beam position is within 10 degrees from the first irradiation axis.

[0024] In one example, a charged particle beam is directed to a sample to form an irradiation region in a sample plane, and a first radiation from the detection region of the sample is detected with 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 is less than a second number of the plurality of detection pixels arranged along the second detector axis. The irradiation region and the detection region are moved in a first direction in the sample plane, and a second radiation from the detection region is detected with a plurality of detection pixels. A first detection axis of the detection region corresponds to the first detector axis, and a second detection axis of the detection region corresponds to the second detector axis, and an angle between the first direction and the first detection axis is less than 45 degrees. A sample image is reconstructed based on the first radiation and the second radiation. In some examples, after the second radiation is detected, the irradiation region and the detection region are moved in a second direction, and a third radiation from the detection region is detected. The second direction may be determined based on sample / system drift. In some examples, the charged particle beam may be adjusted based on sample / system drift while the irradiation region is moved. The second direction may be less than 45 degrees from the first direction. The sample image is further reconstructed based on the third detected radiation.

[0025] 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 examples, the first detection axis is substantially aligned with or parallel to the first direction.

[0026] The charged particle beam or the irradiation area can be scanned / moved substantially along the first irradiation axis by adjusting one or more beam deflection coils that direct the charged particle beam toward the sample and / or a beam limiting aperture in the beam path of the charged particle beam. When the irradiation area (or charged particle beam) is scanned in the first direction, the relative position of the detection area with respect to the irradiation area does not change. That is, the detection area is scanned in the sample plane at the same speed as the irradiation area.

[0027] In some examples, the scanning path of the irradiated area may deviate slightly from the first direction, for example due to factors including the irradiation of the electron beam, the orientation of the detector, and the sample / aperture structure. The sample image can be reconstructed based on the scanning direction and scanning speed of the irradiated area. In some examples, the drift or deviation from the scanning path can be monitored during scanning, and the position of the irradiated area in the sample plane, and / or the first direction (e.g., scanning direction), and / or the optical components for generating the charged particle beam can be adjusted based on the drift. The drift can be detected, for example, based on a reconstructed image of a portion of the sample.

[0028] The sample image can be reconstructed based on the time information of the received radiation. For example, the received radiation can be spatially resolved along the first irradiation axis based on the time when the radiation is detected by the detector. In some examples, the radiated particles are temporally sampled using event-based detection. The sample image can be further reconstructed based on information about the movement of the charged particle beam. For example, the sample image can be reconstructed based on the direction of the irradiated area and the scanning speed. The sample image can be further reconstructed based on the information of the detected radiation. For example, the sample image can be further reconstructed based on the speed of the detected electrons. In some examples, the sample image is reconstructed based on the scanning speed, the scanning direction, and the time of the detected event.

[0029] In some embodiments, one or more passive pixels can be positioned between multiple detection pixels for detecting radiation from the detection region. A passive pixel can be a defective pixel in a 2D detector array. In some examples, one or more empty spaces can be positioned between multiple detection pixels. Because the irradiation region is scanned over the sample and an image is reconstructed based on temporal information of radiation received from different portions of the pixelated array, the methods disclosed herein are less sensitive to defective pixels in the detector array.

[0030] In this way, the number of detection pixels used to detect radiation can be reduced without sacrificing the quality performance of the detector. Compared to a DED with a large field of view, in the present invention, a detector with a lower number of detection pixels can be used, and the same high-quality image of a radiation-sensitive sample can be acquired with a cheaper detector with a lower number of detection pixels. In some examples, the number of detection pixels along the second detector axis can be 100 to 200 times the number of detection pixels along the first detector axis. For example, the number of detection pixels along the first detector axis can be 2, and along the second detector axis can be 128 to 1024. In addition, the spatial resolution of the detector along the first detector axis can be much lower than that of the second detector axis. By reducing the requirements on the detector, the cost of the detector can be further reduced.

[0031] In some examples, the first detector axis and the second detector axis are orthogonal to each other. The first detection axis and the second detection axis are orthogonal to each other. The irradiation region can be formed by a beam limiting aperture positioned in the beam path of the charged particle beam. An example of forming the irradiation region is disclosed in U.S. Pat. No. 11430633 B2, the entire contents of which are incorporated herein by reference and for all purposes.

[0032] 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.

[0033] The detection region is part or all of the irradiation region, where radiation from the detection region is captured by the detector and used to form an image of the sample. In some examples, the detection region is smaller and within the irradiation region. By having an irradiation region that is larger than the detection region, the irradiation region can be extended to overlap with the conductive material surrounding the sample region and avoid charge accumulation in the sample region.

[0034] The detection area may be defined by a first detection edge and a second detection edge parallel to the second detection axis and a third detection edge and a fourth detection edge parallel to the first detection axis. The irradiation area may be defined by a first irradiation edge and a second irradiation edge and a third irradiation edge and a fourth irradiation edge oriented along a first direction. The first detection edge and the first irradiation edge are arranged toward the first direction relative to the second detection edge and the second irradiation edge.

[0035] In some examples, one or more of the detection edge and the irradiation edge can be straight. In some examples, one or more of the detection edge and the irradiation edge can be curved. The position of the detection region relative to the irradiation region can be adjusted by adjusting one or more image deflection coils positioned between the samples and / or adjusting the position and orientation of the detector. In some embodiments, one or both of the irradiation region and the detection region are rectangular.

[0036] During at least part of the scanning and data collection process, portions of the irradiated area can overlap with the conductive material surrounding the sample area. In this way, charge can be removed from the sample during scanning. In one example, the sample is vitrified in a sample area surrounded by a support material (such as carbon). The support material surrounding the sample area can be conductive. The sample area can be any shape. For example, the sample area can be rectangular, square, or circular. In one example, the sample area can be a hole of a TEM grid.

[0037] The detection region may be offset relative to the irradiation region. In other words, at least one of the central axes of the detection region is not aligned with the central axis of the irradiation region. The detection region may be offset relative to the irradiation region toward a scanning direction (e.g., a first direction). In some examples, at least one of the central axes of the detection region parallel and orthogonal to the first direction is not aligned with the corresponding central axes of the irradiation region parallel and orthogonal to the first direction. In one example, the detection region is offset relative to the irradiation region toward the scanning direction. By offsetting the detection region toward the scanning direction, the irradiation region can extend and overlap with materials opposite to the scanning direction, thereby reducing potential radiation damage to unscanned sample areas. In one example, a first distance between a first detection edge and a first irradiation edge is less than a second distance between a second detection edge and a second irradiation edge. The first distance may be non-zero and is determined based on a scanning speed along the first axis and an estimated flux of the received radiation. By keeping the first distance non-zero, initial radiation generated from a particular irradiation region during the scan is not collected. This is because these initial radiations introduce beam-induced particle movement and may reduce the quality of the received signal. One of the third detection edge and the fourth detection edge may substantially overlap with one of the third irradiation edge and the fourth irradiation edge.

[0038] In one example, after scanning along a first direction, data from a first column of a sample area is collected. The irradiated area is then shifted to collect data from a second column of the sample area. The first column and the second column in the sample area are parallel and may or may not overlap. The collected data may be selected for image reconstruction during post-processing. In order to collect data from the second column, the irradiated area may be shifted in a second direction by shifting the charged particle beam by adjusting the beam deflection coils and / or by moving / shifting the sample by adjusting the sample platform. The irradiated area may also be moved by moving an aperture positioned in the charged particle beam path and upstream of the sample plane. When scanning the irradiated area, a post-beam correction is applied to adjust the relative position between the irradiated area and the detection area.

[0039] In this manner, data from most sample areas can be collected, thereby achieving efficient, effective, and high-throughput data collection.

[0040] Steering Figure 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 can displace or move the area irradiated by the electron beam (i.e., the irradiation area) in a sample plane 110 (i.e., an XY plane). The deflected beam enters an upper opening 107 of an optional cryogenic box 108 through an upper objective lens pole piece 106. The electron beam entering through the upper opening 107 irradiates a sample (not shown) positioned at one end of a sample holder 109. The beam profile at the sample plane forming the irradiation area 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 platform 102. For example, the sample can be displaced in the sample plane via the sample holder by operating the platform 102. Radiation 120 from the sample leaves the cryogenic box 108 via the lower opening 112 of the cryogenic box. Radiation 120 passes through lower objective lens pole piece 113 and is deflected by image deflection coil 114 in a descanning mode before reaching detector 115. Detector 115 may be a pixelated detector having a plurality of detection elements for receiving radiation. Figure 3C An example arrangement of multiple detection elements is shown. 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.

[0041] The signal from the detector 115 is transmitted to the controller 117 along the control line (bus) to process and form a sample image. The sample image can be displayed on a display unit (not shown). Such processing may include operations such as combination, integration, subtraction, false coloring, edge enhancement and other processing known to technicians. The controller may include a processor 118 and a non-transitory memory 119 for storing computer-readable instructions. The method disclosed herein can be implemented by executing computer-readable instructions stored in the non-transitory memory 119 in the processor 118. For example, the controller can control the microscope to guide the electron beam to the sample, collect data, and process the collected data. The controller can adjust the electron beam energy, position, irradiation area, and intensity by adjusting one or more lenses and / or electron sources. The controller can adjust the sample position via the sample holder and the platform. The controller can adjust the position of the irradiation area via the image deflection coil. The controller can adjust the sample area to be imaged (e.g., the position of the detection area) via the image deflection coil.

[0042] 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.

[0043] Figure 3A and Figure 3B Two example arrangements of irradiation and detection areas are shown. Figure 3C shows that it can be used to detect Figure 3A and Figure 3B An example arrangement of detection elements of a detector of radiation of the detection area is shown in . In these examples, both the irradiation area and the detection area are rectangular.

[0044] exist Figure 3A and Figure 3B In, the irradiation region 320 is the region irradiated by the charged particle beam in the sample plane. Coordinate system 380 shows the coordinates of the irradiation region, and coordinate system 381 shows the coordinates of the detection region. In this article, the irradiation coordinates and the detection coordinates are the same. The Y axis is the first irradiation axis, and the X axis is the second irradiation axis. The irradiation region is scanned in the sample plane along the first irradiation axis (Y axis of 380, 381) in the direction 350. The detection region 310 is within the irradiation region 320 and is smaller than the irradiation region. The first detection axis is the same as the first irradiation axis, and the second detection axis is the same as the second irradiation axis. The width 304 of the detection region (along the scanning direction 350) is smaller than the width 302 of the irradiation region. The length 301 of the irradiation region can be greater than or the same as the length 303 of the detection region. The irradiation region is bounded by a first irradiation edge 309, a second irradiation edge 313, a third irradiation edge 315, and a fourth irradiation edge 314. The irradiation area has a width 302 and a length 301. The detection area 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 area has a width 304 and a length 303.

[0045] exist Figure 3A and Figure 3B In both, the detection region 310 is offset from the irradiation region 320 toward the scanning direction 350. The central axis 308 of the detection region 310 does not overlap with the central axis 305 of the irradiation region 320. The distance between the first irradiation edge 309 and the first detection edge 307 is less than the distance between the second irradiation edge 316 and the second detection edge 313. In this way, the irradiation region 320 can extend in a direction opposite to the scanning direction 350 (opposite to the direction of the Y axis) to cover a portion of the material surrounding the sample area and promote charge removal. In addition, the distance between the first irradiation edge 309 and the first detection edge 307 can be greater than zero so that the initial radiation generated in response to the irradiation is not detected by the detector.

[0046] Herein, the central axis 321 of the detection region 310 does not overlap with the central axis 306 of the irradiation region 320. The detection region 310 may be offset in different directions along the X-axis, such as Figure 3A and Figure 3BIn other words, the distance between the third irradiation edge 315 and the third detection edge 318 may be less than ( Figure 3A ) or greater than ( Figure 3B ) The distance between the fourth irradiation edge 314 and the fourth detection edge 317. The direction in which the detection area is offset from the irradiation area can be determined based on which portion of the sample area is scanned so that the irradiation area can extend to cover the material surrounding the sample area, and the sample area that has been scanned. In some examples, the third irradiation edge 315 can overlap with the third detection edge 318, and / or the fourth irradiation edge 314 can overlap with the fourth detection edge 317.

[0047] Figure 3C shows the method for receiving / getting from Figure 3A and Figure 3B 381 . An example arrangement of multiple detection elements (or pixels) of a pixelated detector for detecting radiation in a detection area in FIG. Coordinate system 382 is a coordinate system of the detector. The detection 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 detection axis (Y axis of 381), and the second detector axis corresponds to the second detection axis (X axis of 381). The detection elements (e.g., 330 to 337) arranged along the second detector axis can detect spatially resolved radiation along the second detection axis. The detection elements (e.g., 330, 341, 342, and 343) arranged along the first detector axis can detect spatially resolved radiation along the first detection axis. The number of detection elements along the first detector axis is less than the number of detection elements along the second detector axis. The shape of each detection element can be square or rectangular. In some examples, the detection elements can be other shapes. The spatial resolution of each detection element along the second detection axis can be higher than that of the first detection axis. In one example, the first detector axis is aligned with the scanning direction 350. In some examples, Figure 3C The detection elements shown in may be a subset of the detection elements of the detector.

[0048] In some embodiments, the detection elements are arranged in a 1D array. In other words, the number of detection elements along the first detection axis is one. In some embodiments, the irradiation area may be other shapes, such as circular, square, or elliptical.

[0049] In some embodiments, the scanning direction may not be aligned with the detection axis or the irradiation axis. For example, the irradiation area 320 may be scanned along a direction that makes a non-zero angle with the direction 350. In some embodiments, the detection axis may make an angle of less than 45 degrees with the scanning direction.

[0050] Figure 3DAnother example arrangement of an irradiation region 351 and a detection region 352 is shown. The detection region 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 is not aligned with the irradiation coordinate system 380. The scanning direction 350 is neither aligned with the first detection axis (the Y axis of the detection coordinate system 381) nor with the first irradiation axis (the Y axis of the irradiation coordinate system 381). The first detection axis (the Y axis of the detection coordinate system 381) is at an angle 355 with the scanning direction 350. The angle 355 is less than 45 degrees.

[0051] 4A to 4H An example scanning scheme for collecting data from a circular sample area is shown. In this context, the sample area 401 is a 2 μm diameter hole of a TEM grid. The sample (e.g., including a plurality of particles) can be vitrified within the hole and the sample can be visualized using Figure 1 TEM system imaging. FIG. 4A to FIG. 4E Sequentially, the left side of the sample area 401 (eg, the left side relative to the central axis 409) is scanned with the irradiation region 402, and data is collected from the corresponding detection region 403. FIG. 4F to FIG. 4H In sequence, the right side of the sample area (eg, the right side relative to the central axis 409) is scanned with the irradiation region 410, and data is collected from the corresponding detection region 411. The relative arrangement of the irradiation region and the detection region may be similar to FIG. 3A to FIG. 3B Same as shown in .

[0052] exist Figure 4A In FIG. 4 , at the initial first beam position, the irradiation region 402 is positioned at the upper left corner of the sample area 401. Figure 4B At the next beam position shown, the irradiation area is moved in the scanning direction along the first irradiation axis (Y axis) by shifting the beam. As the irradiation area is further scanned along the first irradiation axis, sample data is collected from the first column 406 of the sample area. The sample image generated based on the data collected from the first column 406 is shown in FIG. Figure 4C At each of the beam positions used to scan the first column, the irradiated area overlaps with a region outside the sample area 401 to avoid charge accumulation.

[0053] exist Figure 4D At , the irradiation area 402 is shifted along the second irradiation axis (X axis) toward the right side of the first column. In addition, the irradiation area 402 is shifted back along the first irradiation axis to start collecting data from the second column 407 of the sample area. FIG. 4C to FIG. 4D The displacement can be accomplished by beam displacement via beam deflection coils and / or sample displacement via a sample holder. Additionally or alternatively, the displacement can be accomplished by adjusting the beam limiting aperture. Figure 4E At 404, the irradiation area 402 is scanned along the scanning direction (Y-axis direction) and completes the scanning of the second column 407. In some examples, the second column 407 can be obtained by scanning the irradiation area in a direction opposite to the Y-axis direction. In this case, the detection area is offset relative to the irradiation area toward the scanning direction (opposite to the Y-axis direction).

[0054] The relative position of the detection area 403 with respect to the irradiation area 402 is shown in Figure 3A In the figure, the detection area is offset toward the unscanned sample area (right side). In this way, radiation damage to the unscanned sample area can be minimized because the non-overlapping area between the irradiation area and the detection area is mostly in the scanned sample area.

[0055] exist Figure 4F , the irradiation region 410 is shifted toward the right side of the first column along the second irradiation axis (X axis) compared to the position of the irradiation region 402. In addition, the irradiation region 410 is shifted back along the first irradiation axis to begin collecting data from the third column 412 of the sample area. At the same time, the relative position of the detection region 411 with respect to the irradiation region 410 is different from FIG. 4A to FIG. 4E The relative positions shown in Figure 4E In , the detection area is offset toward the left side of the irradiation area, as shown in Figure 3B As shown. In this way, the irradiation area 410 can overlap with the area surrounding the sample area 401, and the radiation damage to the unscanned sample area can also be reduced. The irradiation area 410 is scanned in the scanning direction along the first irradiation axis, and the data collection of the third column 412 is completed, as shown. Figure 4G Then the irradiation area is shifted along the second irradiation axis and scanned along the first irradiation axis to complete the data collection of the fourth column 413, as shown in FIG. Figure 4H shown.

[0056] Although 4A to 4H The beam positions are shown as discrete positions, but the scanning along the first irradiation direction may be performed continuously. That is, the irradiation area is continuously moved or scanned along the first irradiation axis at a non-zero scanning speed.

[0057] Figure 5 is a combined view of the locations of the irradiation and detection regions during a scan of the sample area 510. 4A to 4H , two relative positions between the irradiation area and the detection area are used to scan different sides of the sample area. 4A to 4H Differently, the sample area is covered by six columns (ie, six scans along the first irradiation direction).

[0058] Figure 6 yes Figure 5602 does not overlap with the first irradiation edge 601. As such, the first few electrons generated in the sample region between the first detection edge 602 and the first irradiation edge 601 are not detected. The distance between the two edges may be arranged based on the scanning speed along the first axis and the estimated flux of the received radiation.

[0059] Figure 7 The use of a charged particle microscope such as Figure 1 Method 700 for imaging a sample using a TEM system (as shown).

[0060] At 702, a sample is loaded into a charged particle microscope and parameters for imaging the sample are set. In one example, the sample is a vitrified sample positioned on a TEM grid. The sample can be loaded into the microscope using a sample holder. Loading the sample can include positioning the sample in a sample plane and in a beam path. In some examples, one or more sample images can be acquired to assist in positioning the sample. Parameters for imaging the sample can include one or more of a beam current, a scan path, a scan speed, the size of an irradiation region and a detection region, and the position of a detection region relative to the irradiation region. The parameters can be determined based on the sample type. For example, based on user input of the sample type, the scan speed can be determined based on the dose limit of the sample type. The flux of radiation received from the sample can be estimated based on the sample type and the sample distribution.

[0061] In one example, the size and shape of the irradiation region can be selected based on one or more of a region of interest (ROI) to be imaged on the sample, a dose constraint, and a scan speed. The ROI can be any shape, such as a circle or a rectangle. Additionally, the position of the detection region relative to the irradiation region can be determined. In some examples, the size of one or both of the irradiation region and the detection region can change during the scan. In some examples, the relative position of the detection region relative to the irradiation region remains the same throughout the scan. The position of the detection region relative to the irradiation region can 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 a first detection edge and a first irradiation edge can increase with the scan speed and decrease with an increase in the estimated flux. As another example, the offset of the detection region relative to the irradiation region can depend on the scan path, such as 4A to 4H In yet another example, the scan direction may vary 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 tune one or more of the imaging parameters before imaging the sample of interest.

[0062] At 703, the charged particle beam is directed towards the sample via beam deflection coils and forms an irradiation region in the sample plane.Step 703 may include selecting a position of the detection region relative to the irradiation region from 702 and determining corresponding parameters for the beam deflection coils and image deflection coils.

[0063] At 704, the irradiation area is scanned in a first direction along a first irradiation axis in a sample plane according to the parameters set at 702, and radiation from the detection area is collected by a detector. The scan may be a step-by-step 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. The drift may include system drift and sample drift. To correct for drift, the scan path or the position of the irradiation area may be adjusted based on the estimated drift.

[0064] At 706, the method 700 checks whether data acquisition along the first irradiation axis is complete (ie, whether data collection along one column is complete). If the answer is yes, the method 700 moves to 708. Otherwise, scanning continues in the first direction.

[0065] At 708, method 700 checks whether data collection from the ROI is complete. If the answer is yes, then at 710, one or more sample images are reconstructed based on the collected data. If data collection from the ROI is not complete, then at 712, the irradiation region is repositioned on the sample plane to continue data collection for another area in the ROI. For example, at 712, the irradiation region may be shifted along a second irradiation axis to collect data from another column of the ROI. The position of the detection region relative to the irradiation region may also be changed at step 703 to collect radiation corresponding to the repositioned irradiation region.

[0066] The technical effect of scanning the irradiation area and acquiring radiation from the detection area, and detecting the radiation via the detector is that the cost of the detector can be reduced while obtaining a sample image with high spatial resolution, wherein a first number of multiple detection pixels arranged along the first detector axis is lower than a second number of multiple detection pixels arranged along the second detector axis. By using a special detection area shape (formed by an aperture), the method enables efficient sample area sampling and reduced sample damage. In addition, by scanning the irradiation area, drift correction can be applied via real-time feedback. Compared with standard methods, this method can scan a larger FOV. The technical effect of making the detection area within the irradiation area and smaller than the irradiation area is that the irradiation area can cover the material surrounding the sample area for charge removal. The technical effect of constructing the sample image based on the time information of the received radiation and the scanning speed is that the spatial resolution requirement of the detector along the first detector axis is lower. In addition, the beam can be scanned continuously along the scanning direction, thereby reducing the overall data acquisition duration. In addition, this method is less sensitive to the number of defective pixels in the detector array and illumination defects.

Claims

1. A method for imaging a sample, the method comprising: directing a charged particle beam toward the sample and forming an irradiation region in a sample plane; scanning the irradiation region substantially in a first direction in the sample plane, and detecting radiation from the detection region with a plurality of detection pixels of a detector, wherein a first detector axis of the detector corresponds to a first detection axis of the detection region, and a second detector axis of the detector corresponds to a second detection axis of the detection region, wherein a first number of the plurality of detection pixels arranged along the first detector axis is lower than a second number of the plurality of detection pixels arranged along the second detector axis, and wherein the first detection axis is oriented at an angle less than 45 degrees to the first direction; as well as An image of a sample is reconstructed based on the radiation detected during the scan. 2 . The method of claim 1 , wherein scanning the irradiation region substantially in the first direction comprises displacing the charged particle beam substantially in the first direction relative to the sample.

3. The method of claim 1, wherein when the irradiation area is scanned substantially in the first direction, the size, shape and relative position of the irradiation area and the detection area remain the same. The method of claim 1 , wherein the detection area is within the irradiation area and is offset relative to the irradiation area toward the first direction.

5. The method according to claim 4, wherein the detection area is defined by a first detection edge and a second detection edge parallel to the second detection axis, the irradiation area is defined by a first irradiation edge and a second irradiation edge orthogonal to the first direction, the first detection edge and the first irradiation edge are arranged toward the first direction relative to the second detection edge and the second irradiation edge, and wherein a first distance between the first detection edge and the first irradiation edge is less than a second distance between the second detection edge and the second irradiation edge.

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

7. A method according to claim 4, wherein the detection area is defined by a third detection edge and a fourth detection edge parallel to the first detection axis, the irradiation area is defined by a third irradiation edge and a fourth irradiation edge oriented along the first direction, and wherein one of the third detection edge and the fourth detection edge substantially overlaps with one of the third irradiation edge and the fourth irradiation edge.

8. The method according to claim 1, further comprising: after scanning the sample substantially along the first direction for a first distance, displacing the irradiation region in a second, different direction for a second distance; and scanning the displaced irradiation region substantially in the first direction, and detecting radiation from the detection region.

9. The method of claim 8, further comprising adjusting a position of the detection region relative to the irradiation region prior to scanning the shifted irradiation region substantially in the first direction.

10. The method of claim 1 , wherein scanning an irradiation region in the first direction in the sample plane comprises continuously moving the irradiation region in the first direction, and wherein reconstructing a sample image based on the radiation detected during the scanning comprises reconstructing a position of the detected radiation along the first detection axis based on time information of the detected radiation and a speed at which the irradiation region is scanned in the first direction.

11. A method for imaging a sample, the method comprising: directing a charged particle beam to the sample to form an irradiation region in a sample plane, and detecting first radiation from the detection region of the sample with a plurality of detection pixels of a detector arranged along a first detector axis and a second detector axis, wherein a first number of the plurality of detection pixels arranged along the first detector axis is lower than a second number of the plurality of detection pixels arranged along the second detector axis; moving the irradiation region and the detection region in a first direction in the sample plane, and detecting second radiation from the detection region with the plurality of detection pixels, wherein a first detection axis of the detection region corresponds to the first detector axis and a second detection axis of the detection region corresponds to the second detector axis, and wherein an angle between the first direction and the first detection axis is less than 45 degrees; as well as An image of a sample is reconstructed based on the first radiation and the second radiation.

12. The method of claim 11, wherein the position of the detection zone relative to the irradiation zone remains the same.

13. The method according to claim 12, further comprising: After detecting the second radiation, moving the irradiation region and the detection region in a second direction, and detecting third radiation from the detection region; And further reconstructing the sample image based on the third radiation. The method of claim 13 , wherein an angle between the first direction and the second direction is less than 45 degrees.

15. The method of claim 11, wherein reconstructing a sample image based on the first radiation and the second radiation comprises reconstructing a signal along the first detection axis based on a time when the radiation was detected and a position of the detection region along the first detection axis at the time.

16. 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 toward the sample plane and forming an irradiation area in the sample plane; a pixelated detector for detecting radiation generated from a detection region of the sample in response to irradiating the sample with the charged particle beam, wherein a first detector axis of the detector corresponds to a first detection axis of the detection region and a second detector axis of the detector corresponds to a second detection axis of the detection region; and A controller comprising a processor and a non-transitory memory for storing computer-readable instructions, wherein by executing the computer-readable instructions in the processor, the charged-particle microscope is configured to: scanning the irradiation region via the charged particle column in the sample plane substantially in a first direction; detecting radiation from the detection region via 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 wherein the first direction is at an angle of less than 45 degrees to the first detection axis; as well as An image of a sample is reconstructed based on the radiation detected during the scan.

17. A charged particle microscope according to claim 16, wherein the charged particle column guides the charged particle beam toward the sample plane via one or more beam deflection coils and beam limiting apertures within the charged particle column, and wherein the charged particle column scans the irradiation area by adjusting the beam deflection coils and / or the beam limiting apertures.

18. A charged particle microscope according to claim 16, further comprising one or more image deflection coils for directing radiation from the detection area toward the detector, wherein the relative position of the detection area within the irradiation area is adjusted by the one or more image deflection coils.

19. The charged particle microscope of claim 16, wherein the first direction is aligned with the first detection axis.

20. The charged particle microscope of claim 16, wherein the charged particle microscope comprises a transmission electron microscope system.

21. The charged particle microscope of claim 16, wherein the detector comprises a first number of pixels, and wherein detecting radiation from the detection region comprises detecting the radiation from the detection region using a second, lower number of the pixels of the detector.

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