Distributed imaging allowing sample relaxation

Abstract

A distributed imaging that allows for sample relaxation. CPB images are acquired with pulsed exposures of a field of view (FOV) defined in a region of interest of a sample. Multiple exposures are configured with time intervals greater than a phonon lifetime to reduce sample damage caused by CPB exposures. The FOV can be defined to be spaced apart to control effective irradiation dose based on a combination of direct and evanescent exposures.

Description

Technical Field

[0001] This application relates to charged particle beam imaging with reduced sample damage. Background Technology

[0002] Many samples of interest in electron microscopy imaging are beam-sensitive and are damaged or otherwise altered in response to electron beam exposure. Such alterations can significantly change sample properties, limiting the usefulness of subsequent imaging. Damage can be associated with, for example, the emission or displacement of atoms, the emission of secondary electrons, or bond breaking in response to the electron beam. While damage can be mitigated to some extent by using very low beam doses, low doses generally do not provide satisfactory image quality. Therefore, there is a need for methods that can reduce or eliminate the effects of such damage in sample images while still providing a sufficient image signal-to-noise ratio. Summary of the Invention

[0003] Methods and apparatus are disclosed that provide repeated exposure of a sample region to a charged particle beam (CPB) such that effective exposure is controlled to avoid or reduce CPB-induced sample damage. CPB exposures can be time-spaced, allowing subsequent exposure of the sample region after reversible CPB-induced sample changes due to previous CPB exposures have dissipated or relaxed. This relaxation ensures that the sample region is effectively exposed only to the currently applied CPB, thus avoiding the effects of combinations with previous exposures. Furthermore, CPB exposures can be time- or spatially spaced to avoid the effects of combining current CPB exposure of a selected sample region with fading exposure from previously exposed sample regions. According to the disclosed methods, the total charge, such as the number of electrons applied to the sample region, can be controlled to as little as one. Such limited exposure may be particularly useful for samples such as frozen samples of biological material.

[0004] The foregoing and other features and advantages will become more apparent from the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0005] Figure 1 A representative CPB imaging system is illustrated, providing pulsed CPB exposures within multiple FOVs defined in the ROI of a sample.

[0006] Figure 2 An example is shown of a set of FOVs arranged in a rectangular array of spaced-out FOVs and the corresponding FOV images in response to pulsed CPB exposure. Figure 2 The image above illustrates a specific sequence of FOV image acquisition.

[0007] Figure 3A to Figure 3BAn example is shown: a first rectangular array and a second rectangular array of spaced-out FOVs, wherein the first and second groups are offset along an axis.

[0008] Figure 4 A representative CPB imaging system is illustrated, which provides pulsed CPB exposure of multiple FOVs by deflecting the CPB to each FOV in the FOV and deflecting the associated FOV image bundle along the axis of the CPB detector.

[0009] Figure 5A A representative FOV and the surrounding receding area are shown.

[0010] Figure 5B Examples include, for example Figure 5A The FOVs shown are FOVs with partially overlapping peripheral vanishing regions when exposed to CPB pulses.

[0011] Figure 5C The changes in the surrounding fade-in region after exposure to CPB are illustrated.

[0012] Figure 6 A representative method for CPB imaging using independently exposed FOV is illustrated.

[0013] Figure 7A An example is shown that includes spaced-out FOVs containing the corresponding features of interest.

[0014] Figure 7B Examples are shown as follows Figure 7A The FOV shown is covered by pixels of an array detector positioned to image the FOV.

[0015] Figure 8 Examples of independently exposed FOVs with a fading perimeter and using different doses are shown.

[0016] Figure 9A An example is shown of adjacent square FOVs that are not separated by a fading perimeter region.

[0017] Figure 9B Overlapping exposure with controlled doses of FOV is illustrated.

[0018] Figure 10 A representative computing environment suitable for controlling FOV exposure and image processing is illustrated. Detailed Implementation

[0019] Introduction and Terminology The CPB imaging apparatus and methods disclosed herein allow for imaging of samples with reduced contributions to reversible sample alterations in response to CPB exposure, or without such contributions. In an example, a sample region is exposed to a CPB pulse, wherein the pulse timing is selected to allow recovery of reversible alterations in response to a previous CPB exposure. Alternatively, the CPB exposure can be arranged such that multiple sample regions are exposed, wherein the sample regions are separated by peripheral distances associated with evanescent coupling from the exposed regions. The sample can be held at cryogenic temperatures, and the CPB exposure is arranged to avoid or reduce irreversible sample alterations.

[0020] Many types of samples are beam-sensitive and respond to greater than 40 electrons / Å. 2 While some samples may be damaged, destroyed, or altered by the electron beam dose, others, kept at lower temperatures (typically cryogenic temperatures below 100 K, such as at or near the boiling point of liquid nitrogen at 77 K), can withstand two or more times the dose. Dose-dependent sample alterations include physical, chemical, and other changes that may render post-exposure sample imaging uninformative.

[0021] As used herein, the field of view (FOV) is the region of the sample of interest exposed to a single pulse of charged particle beam (CPB). An image of the sample of interest is obtained by exposing one or more FOVs with multiple CPB pulses. As discussed below, the FOV can be repeatedly or multiple times exposed to CPB to improve the signal-to-noise ratio in the FOV image. The FOV region can be selected, for example, by selecting the FOV region based on the available pulsed CPB beam current, such that the CPB dose associated with the CPB pulse does not produce unacceptable changes in the features of interest of the sample. In some cases, it is desirable to use a relatively large CPB beam current and a relatively short exposure time to produce a suitable FOV image.

[0022] Some types of exposure-related effects at the field of view (FOV) are irreversible and can depend on the total dose or total exposure energy, while others disappear or diminish over time after exposure. Irreversible changes may be associated with so-called impact displacement, where atoms in the sample are ejected or displaced by exposure. Reversible changes may be associated with the ejection of secondary electrons, sample charging, and, in some cases, bond breaking. However, the methods disclosed herein do not rely on any specific mechanism of reversible or irreversible changes.

[0023] As used in this article, "FOV relaxation time" or "relaxation time" t relaxThe FOV relaxation time is the time interval from the time of exposure to the CPB pulse to the time associated with the recovery of the sample from recoverable changes caused by the CPB exposure. The FOV relaxation time is also associated with the time interval following the CPB exposure, at which subsequent CPB exposures produce changes in the sample independent of the previous CPB exposure, regardless of any irreversible changes caused by the first exposure. The sample can be considered to have returned to its pre-exposure state, and the effect of subsequent CPB exposures is independent of the previous exposure unless the previous exposure reached a level that produced irreversible changes. Relaxation Time t relax It can be correlated with the lifetime of phonons generated by CPB exposure, which is typically between 1 μs and 10 μs.

[0024] FOV exposure to CPB also produces a peripheral region of the FOV, which is referred to herein as the “effectively exposed region” based on the evanescent CPB effect from the associated exposed FOV. Evanescent exposure contrasts with exposure caused by charged particles incident on this region (which may be referred to as “direct” exposure). Effective exposure dissipates with an evanescent time constant that is typically much shorter than the FOV relaxation time. To minimize sample changes in response to CPB exposure, these peripheral regions are considered to have been effectively exposed when the associated FOV is exposed. For this reason, FOVs to be exposed can be spaced out, taking into account the extent of such effectively exposed regions, to avoid applying excessive CPB dose to the peripheral regions. Alternatively, subsequent exposures can be time-spaced based on the evanescent relaxation time. As used herein, the distance associated with evanescent exposure is referred to as… L ev Furthermore, adjacent and sequentially exposed FOVs can be spaced by this distance to avoid or reduce sample alterations caused by CPB. Evanescent exposure tends to be more efficient than relaxation time. t relax The decay is much faster and lasts for less than 1 ns, so fading exposure is typically considered only for FOVs that are sequentially exposed over a period of less than 5 to 10 ns. (Evanishing distance) L ev It changes over time after exposure and rapidly decays to zero.

[0025] "Dose" refers to the charge / area or energy / area associated with CPB exposure. The sample response to any CPB dose varies with the total charge (the product of the CPB pulse duration and the CPB beam current), CPB beam energy, CPB current, and CPB pulse duration. For ease of description, the change in sample response to CPB exposure is referred to as dose dependence.

[0026] In some examples, FOV exposure and associated images are paired with a "timestamp" or other indicators that allow the FOV images to be assembled into a larger image of the sample ROI. For FOVs obtained via raster scanning of the CPB, FOV image sequences can be assembled based on the number of FOVs in a row and the number of scan rows, and no additional location indicators are required. In some cases, the FOVs are arbitrarily chosen, and indications of the CPB (and FOVs) at a specific time are required.

[0027] A beam or radiation beam refers to a propagating charged particle or propagating electromagnetic radiation, whether collimated or uncollimated. In some examples, the field of view (FOV) is arranged as a rectangular array of rows and columns. Arrangements described relative to columns or rows can be similarly provided using columns and rows respectively.

[0028] As used herein, an FOV image beam refers to radiation exposed to the CPB in response to an FOV, which can be focused to form an image of the FOV at the detector. Such an FOV image beam can be focused or unfocused at various locations within the CPB optical system and can be referred to as propagating along the axis, regardless of whether it is focused or not.

[0029] Various FOV shapes and sizes can be used, and the CPB pulse duration can be varied. The FOV shape and size can differ for each FOV, and the dose applied to each FOV can also vary. CPB pulse durations of less than 1 ns, 10 ns, 100 ns, 1 μs, 10 μs, 100 μs, 1 ms, or others can be used. The number of CPB pulses applied to each FOV can vary, typically ranging from 2 to 10. 9 The range can be greater than or equal to, depending on the beam current and the desired amount of charge to be applied to each pixel area. The FOV size can be a fraction of a nanometer, a few micrometers, or other large or smaller dimensions. The FOV can be square, rectangular, polygonal, elliptical, circular, or any other shape with any combination of curved and linear sides.

[0030] The CPB pulses can be selected to provide as few as 1, 2, 5, 10, or 20 charged particles per pulse, and an acceptable image signal-to-noise ratio can be achieved by combining FOV images from multiple exposures or by averaging these images. In a typical example, the CPB is an electron beam, and the imaging system is an electron microscope.

[0031] In some examples, ultrashort electron pulses comprising no more than 10,000, 1,000, 100, 50, 25, 10, 5, or 2 electrons are applied to avoid sample damage. The electron pulses are shifted to different fields of view (FOVs) using a scanning deflection system, resulting in FOVs that are separated by a factor of 5, 2, 1, 0.5, or 0.25. L evThe distance.

[0032] As used in this application and claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly indicates otherwise. Additionally, the term “comprising” means “including.” Furthermore, the term “coupled” does not exclude the existence of intermediate elements between coupled items.

[0033] The systems, apparatuses, and methods described herein should not be construed as limiting in any way. Rather, this disclosure relates to all novel and non-obvious features and aspects of the various disclosed embodiments, whether individually or in various combinations and sub-combinations formed with each other. The disclosed systems, methods, and apparatuses are not limited to any particular aspect or feature or combination thereof, nor are they required to possess any one or more particular advantages or problems solved. Any operational theory is provided for ease of interpretation, but the disclosed systems, methods, and apparatuses are not limited to such operational theories.

[0034] Although some of the methods disclosed are described in a specific order for ease of presentation, it should be understood that this descriptive approach includes rearrangement unless the specific language described below requires a particular order. For example, operations described sequentially may be rearranged or performed simultaneously in some cases. Furthermore, for simplicity, the accompanying drawings may not show the various ways in which the disclosed systems, methods, and apparatus can be combined with other systems, methods, and apparatuses. Additionally, this description sometimes uses terms such as “produce” and “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual operations performed. The actual operations corresponding to these terms will vary depending on the specific implementation and can be readily identified by one of ordinary skill in the art.

[0035] In some examples, values, procedures, or devices are referred to as “lowest,” “best,” “minimum,” etc. It should be understood that such descriptions are intended to indicate that a choice can be made among many alternative functionalities used, and that such a choice is not necessarily better, smaller, or otherwise preferred than other choices.

[0036] As used herein, “image” means a display view such as a sample or a portion thereof presented on a display device, and stored data that can be used to produce a display image, such as digital data stored in a non-transitory computer-readable medium, such as JPG, TIFF, BMP files or other formats.

[0037] Example 1 refer to Figure 1A representative CPB imaging apparatus 100 includes a pulsed CPB source 102 operable to generate CPB pulses in response to a CPB pulse controller 104. The CPB source 102 directs the pulsed CPB 103 to a field-of-view (FOV) deflector 106 operable to deflect the pulsed CPB 103 to some or all of the FOVs on the sample S. For illustration, the CPB 103 is illustrated as deflecting along axes 111-113 to FOVs (x1, y1), (x2, y2), and (x3, y3), respectively, where the x and y coordinates are defined relative to coordinate system 191. The CPB optical column 108 may include a CPB lens, deflector, astigmatism corrector, aperture, or other CPB optical elements that shape, focus, and direct the deflected CPB to various FOVs. Figure 1 The specific arrangement is merely an example, and the elements of the CPB optical column 108 and the FOV deflector 106 may be arranged in different orders along the CPB optical axis 126.

[0038] CPB imaging lens 120 is positioned to receive radiation beams 151-153 associated with CPB exposures of FOVs 1-FOV 3, and directs these beams to an FOV image deflector 122, which is configured to direct the beams 151-153 along the CPB optical axis 126 to a radiation detector 124. The radiation beams 151-153 may include some or all of the transmitted or scattered portions of the CPB, such as secondary emissions of secondary electrons or photons, and may thus select the radiation detector 124. In some examples, an FOV image deflector is not used, and imaging lens 120 is sufficient to direct the radiation beams 151-153 to the radiation detector 124.

[0039] Controller 130 is coupled to CPB pulse controller 104, FOV selection deflector 106, and FOV image deflector 122 to control the irradiation of selected groups or sequences of FOVs and to direct radiation in response to FOV irradiation to radiation detector 124. Controller 130 may randomly select FOVs as a group of rasterized FOVs, or have FOV separation considering other options such as FOV relaxation time and the effective exposed peripheral area. Typically, a particular FOV is re-irradiated only after a time greater than or equal to the FOV relaxation time has elapsed. If adjacent or neighboring FOVs are to be irradiated sequentially, the controller selects CPB deflection such that the FOVs are separated based on the size of the effective exposed peripheral area. Controller 130 typically assembles FOV images to generate one or more images of ROIs for display on display device 132; however, alternatively or supplementally, ROI images and / or FOV images may be transmitted via a network or other connection for remote display and processing.

[0040] Example 2 refer to Figure 2 The surface of sample 200 defines a region of interest (ROI) 202, which is divided into multiple fields of view (FOVs), exemplified as follows: m × n FOV array FOV ( i , j ),in i =1、…、 m ,and j =1、…、 n ,in m and n It is an integer greater than 0. Region 206 is selected as the reference region and can be used to establish CPB deflection for selecting FOV, measuring CPB beam current, CPB pulse width, or other purposes. FOVs can be irradiated row by row, for example, by irradiating the first row of FOVs FOV(1,1), FOV(1,2), ..., FOV(1,1 ... n ), followed by the second row of FOV: FOV(2,1), FOV(2,2), ..., FOV(2,1), ..., FOV(2,2). n ), with FOV(2,1) or FOV(2, n (Starting from point 250). Therefore, the FOV can be scanned to irradiate in a single direction 250, or irradiation can be performed by alternating scans in directions 250 and 252. Figure 2 In the example, the FOVs in a row are spaced 2 units apart in the X direction of coordinate system 210. L eff ,in L eff This is the effective exposure length associated with the exposure of the FOV. Rows in the FOV array can be similarly spaced. Since the effective exposure length typically decays rapidly, for image acquisition using unidirectional scanning, a scan of an FOV such as FOV(1,1) can be temporally sufficiently separated from scans of adjacent FOVs (2,1), making it possible to perform image acquisition based on... L eff Separating lines is unnecessary.

[0041] Obtain the image corresponding to each FOV. Figure 2 The image shown represents a typical field of view (FOV). I For example, a set of 2701 images. I (1,1) I (1,2), ... I (1, n Associated with the first row of FOV, a set of 2702 images I (2,1) I(2,2), ... I (2, n This is associated with the second row FOV, and a set of 270 m image I ( m ,1) I ( m ,2),...、 I ( m , n ) and the m Row FOV is correlated. Multiple sets of 270 images can be repeatedly acquired to improve image quality, where each exposure in the FOV exposure is temporally separated based on the FOV relaxation time. In one example, the image... I (1,1) I (1,2), ... I ( m , n At the corresponding time t 1. t 2, ... t (m-1)n+1 … t m*n The images are acquired sequentially. This imaging sequence can be repeated until satisfactory images are obtained for some or all of the fields of view (FOVs). Other sequences of image acquisition can be used, such as randomly selecting FOVs, or sequentially along the column direction or along a direction that is neither parallel to the rows nor the columns.

[0042] Example 3 In some cases, spacing out the fields of view (FOVs) may result in inadequate or complete imaging of parts of the area of ​​interest (ROI). The space between FOVs can be shown in Figure 3A to... Figure 3B The example shown is an imaging process. Referring to Figure 3A, as in Figure 2 In the sample 300, the ROI 302 was divided into a first group of 314 FOVs, which were arranged as follows: m × n An array of FOVs, wherein each FOV is spaced apart from FOVs in adjacent rows and / or columns. As shown in Figure 3A, the first column of FOVs (and the first group 314) are offset from the reference position by a distance... L off1 To avoid gaps in the images produced by imaging the FOV of the first group of 314, such as... Figure 3B As shown, the offset distance is limited. L off2The second group of 316 FOVs has FOVs located between corresponding FOVs in the first group of FOVs. In this example, the centers of the FOVs in the second column of the second group of 316 FOVs are aligned on axis 320, which is parallel to the X-axis of coordinate system 310 and equidistant from the FOVs in the first and second columns of the first group of 314 FOVs. Other offsets between the first and second groups can be used, such as distances corresponding to the separation of FOVs in each row. Generally, any offset that provides CPB exposure to all parts of the ROI can be used. Multiple groups of two, three, or more FOVs can be used, and the FOVs in each group do not need to have the same spacing in the X or Y direction or use a common FOV size. Offsets in both row and column directions can be provided, and for illustrative purposes, column offsets are shown.

[0043] It should be understood that the FOVs to be imaged can be arranged in an array other than a rectangular array, or they can be arranged randomly or arbitrarily around the ROI. The FOVs do not need to be spaced out for sufficiently long time intervals between successive irradiations. The FOVs defined by the array do not need to be imaged sequentially. For ease of illustration, [details omitted]. Figure 2 and Figure 3A to Figure 3B Examples. In some cases, it is more convenient to scan the FOV rows in the rectangular array in alternating directions to avoid the delay associated with scanning the CPB back to the first side of the ROI.

[0044] Example 4 refer to Figure 4A representative CPB imaging apparatus 400 includes a CPB source 402 positioned to guide a CPB beam along axis 401. A CPB beam deflector 405 is positioned to selectively deflect the CPB beam to be transmitted or blocked at an aperture plate 406 to generate CPB pulses. In some examples, a pulsed CPB source is used, eliminating the need for such a photoelectric emitting surface combined with a pulsed laser and a CPB beam deflector. An FOV selection deflector 410 receives the pulsed CPB and includes a first deflector 410A and a second deflector 410B. The first deflector generates CPB 408 propagating away from axis 401, while the second deflector redirects the deflected CPB from the first deflector 410A to propagate as a redirected beam 409 parallel to axis 410. The redirected beam 409 is coupled to the FOV on the sample 414 via a first pole piece 411A of a magnetic objective. Sample 414 is held within a housing 413 including CPB apertures 412 and 415 and is thermally coupled to a cold finger 416 to establish a sample temperature, such as a cryogenic temperature, and thermally coupled to a sample stage 418 for positioning relative to axis 401. A radiation beam 417 (such as transmitted charged particles or secondary electrons) in response to a dose applied to the FOV is coupled by a lower electrode 411B to an FOV image beam deflector 420, which includes a first deflector 420A and a second deflector 420B. The first and second deflectors guide the radiation beam 417 to propagate parallel to axis 401 and orthogonally incident, centered on CPB detector 422. Such an FOV image beam deflector is not mandatory but allows for the use of a smaller CPB detector and provides more uniform detection of the radiation beam as the FOV position changes. The selection of CPB deflection, scan rate, current, FOV size, FOV position, FOV shape, exposure delay, repetition rate, and FOV image averaging and combination can be provided by controller 424 (such as a CPU or other processing device) based on processor-executable instructions and associated input and output data, which can be stored in one or more non-transitory processor-readable storage media or devices 426.

[0045] Example 5 Figure 5A Examples of FOVs 502 and 506 with corresponding surrounding areas 504 and 508 associated with CPB exposure elapsed distance are shown. Figure 5AIn the example, surrounding regions 504 and 508 share boundary 509 at some point after CPB exposure. The extent of surrounding regions 504 and 508 decreases over time after CPB exposure. Surrounding regions 504 and 508 are effectively exposed through their respective CPB exposures, and in this example, additional CPB exposure of either FOV does not interact with the exposure of the other FOV to cause damage within the FOV or its corresponding surrounding region. Because the extent of the surrounding regions changes over time, surrounding regions 504 and 508 can have different extents and still share boundary 509. Figure 5B Examples of FOVs 516 and 518 are shown, which have peripheral regions that partially overlap due to weak evanescent coupling, but multiple effective exposures of the FOVs within them are not associated with sample changes. Figure 5C The figure shows the effect of time on CPB exposure. t 1 , t 2 and t 3 The changing area around FOV 522, the surrounding region 524A-524C, is contracting. As shown in the figure, the fading region 524A-524C extends a certain distance. L ev This distance changes over time from the start of CPB exposure.

[0046] Example 6 refer to Figure 6 Representative method 600 includes determining the number of FOVs in the ROI at 602. Typically, FOVs are selected by examining an image of the ROI to identify any features of interest and choosing FOVs for each feature of interest. At 604, the ROI is divided into one or more groups of FOVs, and the exposure order is determined to establish scan rate, effective dose, and FOV separation. At 606, counters are initialized. I And at location 608, the irradiation was... I The first FOV, and the first sample was collected at 610. I Images of the field of view (FOV). If more FOV images are to be acquired at position 612 as determined, the counter is incremented at position 616. I The FOVs are then irradiated and imaged. Each FOV can be exposed once or multiple times to provide satisfactory FOV images. If all FOVs in a set have been imaged, it is determined at 617 whether to acquire additional sets of FOV images to provide an acceptable image signal-to-noise ratio. If additional sets of FOV images are to be acquired, the counter is reinitialized at 606. IThis begins the acquisition of a subsequent set of FOV images. It can be determined at 622 whether to combine FOV images, and if so, at 626 to combine FOV images to form an ROI image larger than the FOV (or an image of a portion of the ROI). Alternatively, in many examples, the FOVs are not adjacent, and a separate FOV image is provided at 624. As discussed above, in most cases, multiple images of each FOV are obtained to provide an average FOV image with a sufficient image signal-to-noise ratio.

[0047] exist Figure 6 In the example, multiple FOVs are imaged one after another, and then repeatedly re-imaged to achieve satisfactory image quality. During the exposure of an FOV, as discussed above, the CPB effect in the previously exposed FOV is reduced. However, each FOV can be repeatedly exposed before switching to another FOV.

[0048] Example 7. ROI Selective imaging In typical practical examples, only the chosen FOV of a much larger ROI is of interest. (Reference) Figure 7A ROI 700 includes FOVs 702, 704, 706, and 708 to be imaged to study features of interest 703, 705, 707, and 709, respectively. The FOVs are shown as rectangular regions that can be selected to be imaged onto pixels of a rectangular detector array based on the magnification of the imaging system. Figure 7A In the examples, FOVs 704 and 706 define an overlapping region 720, but in most cases, such overlapping regions can receive radiation without concern for damage because they do not contain the features of interest or are unlikely to do so for most samples. However, if necessary, CPB exposure can be arranged to allow sufficient relaxation in regions such as these to avoid beam-induced sample damage.

[0049] FOV 704 Figure 7BThe image is magnified and overlaid to indicate the mapping to pixels 713 (such as representative pixels 714) of the detector array. Some pixel sizes of interest are 1Å×1Å, 2Å×2Å, 5Å×5Å, 10Å×10Å, 20Å×20Å, 50Å×50Å, or other sizes. A 4×5 pixel array is illustrated, but M×N arrays are typically used, where M and N are integers, with typical values ​​ranging from 100 to 5000. In some examples, a 2000×2000 array (4M pixels) is used. The CPB, such as the electron beam irradiable region 722, is shown as a circular area for convenience. The electron beam intensity is chosen such that during any electron beam pulse, the area corresponding to the FOV of a pixel does not receive more than a single electron to avoid damage or degradation of the feature of interest. Pulses with single electrons can be used and repeated until the FOV areas associated with all pixels have received enough electrons to provide a sufficient FOV image signal-to-noise ratio. To provide a single electron exposure for each pixel in the pixel array, the maximum number of electrons is equal to the number of pixels; pulses exceeding this will have more than one electron per field of view (FOV) associated with the pixel. Therefore, the number of electrons per pulse ranges from 1 electron / pulse to MN electrons / pulse. However, for electron / pulse numbers close to the upper limit MN, it becomes possible that some FOVs associated with the pixel will receive more than one electron while others will not receive any. The number of electrons / pulses can be selected based on the probability of multiple electrons in a pixel region, and is chosen to maintain spacing between exposed pixels to avoid coupling via fading exposure.

[0050] Charge / pulse ratio is a crucial factor in image setup time. Other factors include relaxation time and the preferred number of electrons associated with each pixel. The imaging time T when using a single-electron pulse... image It is T image = QMN t relax These single-electron pulses are repeatedly and sequentially applied to each pixel in the M×N array, such that each pixel has a relaxation time t relax The response period is to a pulse of irradiation, where repeated pulses associate each pixel with (average) Q charges, where M, N, and Q are non-negative integers. For a 4M pixel detector array and Q = 40 electrons / pixel, where t relax = 10μs, T image =1600s. For each pulse charge at the upper limit of a single charge / pixel (i.e., MN electrons / pulse), each FOV region associated with a pixel receives electrons during each pulse, such that the total imaging time T image = Q t relaxOr use Q = 40 and t relax = 400μs of 10μs. However, at such a high electron / pulse number, many FOV regions associated with a pixel may receive multiple electrons in a single pulse, and a smaller electron / pulse number is generally preferred.

[0051] Refer again Figure 7A Imaging of FOVs 702, 704, 706, and 708 can be accomplished either by sequentially guiding the CPB to each FOV or by imaging the selected FOVs first and then the remaining FOVs. With sequential imaging, the time between imaging each FOV can be used to provide some or all of the relaxation time for subsequent irradiation without damaging the sample.

[0052] Example 8 Figure 8 Examples are given for the first group of 800 individuals exposed to CPB doses D1 and D2, respectively. N FOV and Group 820 N FOV, of which N It is a positive integer. The representative FOVs 804-806 of the first group of 800 have surrounding evanescent regions 814-816; the representative FOVs 824-826 of the second group of 820 have surrounding evanescent regions 834-836. Some or all FOVs may receive different CPB doses, and the FOVs in each row or column of the rectangular array do not need to receive a common dose. For some samples, the selected FOVs may be more or less sensitive to CPB exposure or exposure parameters (such as pulse duration, CPB current, CPB energy, or others), and the CPB pulses may be appropriately (and individually) configured for each FOV. In some examples, the selected FOVs are less important, and fewer CPB pulses are applied to accelerate FOV image acquisition.

[0053] Example 9 Figures 9A to 9B Additional arrangements of the FOV and methods for irradiating and imaging the FOV are illustrated. Figure 9AAn array of FOVs defined within the ROI of sample 902 is illustrated, where FOVs contact adjacent FOVs without any separation based on fading exposure. In this example, FOVs are labeled from 1 to 12 to indicate the order of exposure, such that adjacent FOVs are not exposed immediately before or after any FOV exposure. For example, FOV 910, labeled as the 11th exposure, is adjacent to FOVs 911 and 912, which are the 5th and 6th exposures, respectively, such that no FOV separation is provided depending on the time constant associated with fading exposure. Similarly, FOV 912 is the 6th exposure, which occurs exactly before the exposures of FOVs 911 and 913. Figure 9A The FOV is exemplified as being exposed in a specific order, but other orders can be used, such as random selection of FOV, which is constrained by the time between multiple exposures based on the FOV relaxation time as discussed above.

[0054] Figure 9B An example is illustrated of a set of 960 FOVs defined on substrate 950. In this example, FOVs are defined in columns 952, 956 and 954, 958, where the FOVs of columns 952, 956 overlap with the FOVs of columns 954, 958. In this example, FOV overlap is permitted, but such double exposure of any overlapping area of ​​FOVs, with additional exposure delayed based on FOV-related time, is allowed. Multiple FOV overlaps can be used to allow FOV relaxation based on a suitable CPB exposure delay. In some examples, only the exposure of selected FOVs undergoes a delay based on FOV relaxation, while the FOVs themselves do not undergo this delay.

[0055] Example 10. Representative computing environments Figure 10The following discussion aims to provide a brief overview of an exemplary computing environment in which the disclosed techniques can be implemented. In particular, some or all portions of this computing environment may be used with the methods and apparatus described above to define a controller or control system for, for example, controlling beam deflection, FOV selection, pulse rate, FOV dose, and processing FOV and ROI images. Although not required, the disclosed techniques are described in the general context of computer-executable instructions executed by a personal computer (PC), such as program modules. Typically, program modules include routines, programs, objects, components, data structures, etc., that perform specific tasks or implement specific abstract data types. Furthermore, the techniques of this disclosure can be implemented using other computer system configurations including: handheld devices, tablets, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, microcomputers, mainframes, etc. The disclosed techniques can also be practiced in a distributed computing environment where tasks are performed on remote processing devices linked via a communication network. In a distributed computing environment, program modules may reside in both local and remote memory storage devices. In some cases, such processing is provided remotely, while in others, and more typically, such processing is provided within a dedicated microscope system. The disclosed system can be used to control image acquisition, and provides a user interface as well as act as an image processor.

[0056] refer to Figure 10 An exemplary system for implementing the disclosed technology includes a general-purpose computing device in the form of an exemplary conventional PC 1000, including one or more processing units 1002, system memory 1004, and a system bus 1006 coupling various system components including the system memory 1004 to the one or more processing units 1002. The system bus 1006 can be any of several types of bus architectures, including a memory bus or memory controller, a peripheral device bus, and a local bus using any of various bus architectures. The exemplary system memory 1004 includes read-only memory (ROM) 1008 and random access memory (RAM) 1010. A basic input / output system (BIOS) 1012 is stored in the ROM 1008, and the BIOS includes basic routines that help transfer information between components within the PC 1000.

[0057] The exemplary PC 1000 also includes one or more non-transitory storage devices 1030, such as hard disk drives for reading from and writing to hard disks, disk drives for reading from or writing to removable disks, and optical disk drives for reading from or writing to removable optical disks. Such storage devices may be connected to the system bus 1006 via hard disk drive interfaces, disk drive interfaces, and optical disk drive interfaces, respectively. The drives and their associated computer-readable media provide the PC 1000 with non-volatile storage of computer-readable instructions, data structures, program modules, and other data. Other types of computer-readable media capable of storing data accessible by the PC may also be used in the exemplary operating environment.

[0058] Multiple program modules may be stored in storage device 1030, which includes the operating system, one or more applications, other program modules, and program data. Users can input commands and information to PC 1000 via one or more input devices 1040, such as a keyboard, and pointing devices such as a mouse. For example, a user can input commands to initiate image acquisition or select FOV, ROI, dose, and time constant associated with evanescent and FOB relaxation. These and other input devices are typically connected to one or more processing units 1002 via a serial port interface coupled to system bus 1006, but can also be connected via other interfaces such as a parallel port, Universal Serial Bus (USB), or wired or wireless network connections. Monitor 1046 or other types of display devices are also connected to system bus 1006 via an interface such as a video adapter and can display, for example, a single FOV image, ROI image, or other raw or processed images for FOV and ROI alignment and selection. Other peripheral output devices, such as speakers and printers (not shown), may be included.

[0059] PC 1000 can operate in a networked environment using a logical connection to one or more remote computers (such as remote computer 1060). In some examples, this includes one or more network or communication connections 1050. Remote computer 1060 can be another PC, server, router, network PC, or peer device, or other common network node, and typically includes many or all of the elements described above relative to PC 1000, but... Figure 10 Only memory storage device 1062 is illustrated. Personal computer 1000 and / or remote computer 1060 can be connected to a logical local area network (LAN) and a wide area network (WAN). Such network environments are common in offices, enterprise-wide computer networks, intranets, and the Internet. In some examples, alignment image stacks are transmitted to a remote system for 3D image reconstruction or other processing.

[0060] like Figure 10As shown, the memory 1090 (or a portion of the memory or other memory) stores instructions that can execute the processor to control the CPB deflector, CPB pulse repetition rate, CPB pulse duration, FOV parameters (such as size, shape, separation, FOV dose), and to acquire and assemble FOV images to form a ROI image, as well as store associated exposure data, image data, relaxation time, and elapsed length. FOV images may be stored along with timestamps indicating when the FOV images were acquired (or other indications of the FOV image sequence) so that FOVs can be assembled into an FOV image.

[0061] Public paragraphs Example 1 is a method comprising: applying a plurality of CPB pulses to at least one field of view (FOV) defined in a region of interest (ROI) of a sample, wherein the plurality of CPB pulses are applied to the at least one FOV at a time interval based on the FOV relaxation time; and obtaining a plurality of FOV images of the at least one FOV, each FOV image corresponding to a corresponding CPB pulse among the plurality of CPB pulses.

[0062] Example 2 includes the subject matter described in Example 1, and further specifies that the at least one FOV is defined based on a portion of the image of the ROI containing the features of interest of the sample.

[0063] Example 3 includes the subject matter according to any one of Examples 1 to 2, and further specifies that the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest.

[0064] Example 4 includes the subject matter according to any one of Examples 1 to 3, and further includes combining the FOV images associated with each of the plurality of CPB pulses to produce a combined FOV image.

[0065] Example 5 includes the subject matter according to any one of Examples 1 to 4, wherein the FOV images are combined by averaging or summing to produce the FOV image.

[0066] Example 6 includes the subject matter according to any one of Examples 1 to 5, wherein the plurality of CPB pulses applied to the at least one FOV are temporally separated by at least the FOV relaxation time.

[0067] Example 7 includes the subject matter according to any one of Examples 1 to 6, and further specifies that the FOV relaxation time is the phonon relaxation time.

[0068] Example 8 includes the subject matter according to any one of Examples 1 to 7, and further includes applying a CPB deflection to generate the plurality of CPB pulses.

[0069] Example 9 includes the subject matter according to any one of Examples 1 to 8, and further includes applying an image deflection to a bundle of FOV images associated with the FOV image, the image deflection being selected to guide each of the FOV image bundles along a detector axis.

[0070] Example 10 includes the subject matter according to any one of Examples 1 to 9, and further specifies that the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest, and the method further includes: wherein the image deflection is selected to guide each of the FOV image bundles associated with each of the two or more FOVs along the detector axis.

[0071] Example 11 includes the subject matter according to any one of Examples 1 to 10, and further specifies that the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest, and the image deflection is selected to guide each of the FOV image bundles associated with each of the two or more FOVs to a common detector region.

[0072] Example 12 includes the subject matter according to any one of Examples 1 to 11, and further specifies that each FOV image in the FOV image is generated by an array detector defining a plurality of pixels, wherein the CPB is an electron beam, and each CPB pulse is selected to provide fewer than 2 electrons to the FOV region associated with the corresponding pixel defined by the array detector.

[0073] Example 13 is a method comprising: repeatedly exposing a sample to a field of view (FOV) of a sample to a pulsed plasma pressure (CPB), wherein the CPB pulses are temporally separated by FOV relaxation times associated with recoverable sample damage; and imaging the FOV using an array detector defining a plurality of pixels, wherein the CPB pulses are configured such that the number of charged particles from the CPB pulses in the FOV region corresponding to a pixel in the array detector FOV image associated with each of the CPB pulses is less than 10.

[0074] Example 14 is a CPB device comprising: a CPB source operable to generate a plurality of CPB pulses directed to each of a plurality of fields of view (FOVs) defined on a sample; and a CPB beam deflector positioned to receive the CPB pulses from the CPB source and direct the plurality of CPB pulses to each of the plurality of FOVs, wherein the plurality of CPB pulses applied to each FOV are temporally separated by at least a phonon lifetime associated with the sample.

[0075] Example 15 includes the subject matter described in Example 14, and further includes a CPB image deflector operable to guide a bundle of FOV images associated with each of the plurality of FOVs defined on the sample along a common axis.

[0076] Example 16 includes the subject matter according to any one of Examples 14 to 15, and further includes a CPB image deflector, wherein the CPB image deflector is operable to guide the FOV image beam to a common region of the CPB image detector.

[0077] Example 17 includes the subject matter according to any one of Examples 14 to 16, and further specifies that the CPB is an electron beam, and the CPB device further includes a controller operable to direct the CPB source to generate CPB pulses such that each pixel of the CPB array detector is associated with a corresponding portion of the FOV that receives fewer than 5 electrons in any CPB pulse.

[0078] Example 18 includes the subject matter according to any one of Examples 14 to 17, and further includes a controller operable to combine FOV images based on each FOV image bundle in the FOV image bundle to produce an FOV image.

[0079] Example 19 includes the subject matter according to any one of Examples 14 to 18, and further specifies that the CPB is sequentially deflected to the plurality of FOVs of interest of the sample.

[0080] Example 20 is a method comprising: repeatedly guiding an electron beam to at least one field of view of a sample; and imaging the at least one field of view using an array detector defining a plurality of pixels, wherein the electron beam is configured to effectively expose a pixel-associated FOV region to no more than a selected number of electrons.

[0081] Example 21 includes the subject matter described in Example 20, and further specifies that the electron beam is configured to effectively expose the FOV based on direct electron beam exposure and evanescent exposure.

[0082] Given that the principles of the disclosed technology can be applied to many possible implementations, it should be recognized that the illustrated implementations are merely preferred examples and should not be considered as limiting the scope of this disclosure.

Claims

1. A method, the method comprising: Multiple CPB pulses are applied to at least one field of view (FOV) defined in the region of interest (ROI) of the sample, wherein the multiple CPB pulses are applied to the at least one FOV at a time interval based on the FOV relaxation time; as well as Multiple FOV images are obtained for the at least one FOV, each FOV image corresponding to a corresponding CPB pulse among the multiple CPB pulses.

2. The method of claim 1, wherein the at least one FOV is defined based on a portion of the image of the ROI of the sample containing the feature of interest.

3. The method of claim 1, wherein the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest.

4. The method of claim 1, further comprising combining the FOV images associated with each of the plurality of CPB pulses to generate a combined FOV image.

5. The method of claim 4, wherein the FOV images are combined by averaging or summing to generate the FOV images.

6. The method of claim 1, wherein the plurality of CPB pulses applied to the at least one FOV are temporally separated by at least the FOV relaxation time.

7. The method according to claim 1, wherein the FOV relaxation time is the phonon relaxation time.

8. The method of claim 1, further comprising applying a CPB deflection to generate the plurality of CPB pulses.

9. The method of claim 8, further comprising applying an image deflection to a bundle of FOV images associated with the FOV image, the image deflection being selected to guide each of the FOV image bundles along a detector axis.

10. The method of claim 9, wherein the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest, and further wherein the image deflection is selected to guide each of the FOV image bundles associated with each of the two or more FOVs along the detector axis.

11. The method of claim 9, wherein the at least one FOV is defined as two or more FOVs based on a portion of the image of the ROI of the sample, including the corresponding feature of interest, and further wherein the image deflection is selected to guide each of the FOV image bundles associated with each of the two or more FOVs to a common detector region.

12. The method of claim 1, wherein each FOV image in the FOV image is generated by an array detector defining a plurality of pixels, wherein the CPB is an electron beam, and each CPB pulse in the CPB pulse is selected to provide fewer than two electrons to an FOV region associated with a corresponding pixel defined by the array detector.

13. A method comprising: The FOV on the sample is repeatedly exposed to CPB pulses, wherein the CPB pulses are temporally separated by FOV relaxation times associated with recoverable sample damage; as well as The FOV is imaged using an array detector that defines multiple pixels, wherein the CPB pulses are configured such that the number of charged particles in the FOV region corresponding to each pixel is less than 10 in response to each CPB pulse.

14. A CPB apparatus, the CPB apparatus comprising: A CPB source, which is operable to generate multiple CPB pulses directed to each of a plurality of FOVs defined on the sample; and A CPB beam deflector is configured to receive CPB pulses from the CPB source and direct a plurality of CPB pulses to each of a plurality of FOVs, wherein the plurality of CPB pulses applied to each FOV are temporally separated by at least a phonon lifetime associated with the sample.

15. The CPB apparatus of claim 14, further comprising a CPB image deflector operable to guide a bundle of FOV images associated with each of the plurality of FOVs defined on the sample along a common axis.

16. The CPB apparatus of claim 15, further comprising a CPB image deflector, wherein the CPB image deflector is operable to guide the FOV image beam to a common region of the CPB image detector.

17. The CPB apparatus of claim 14, wherein the CPB is an electron beam, the CPB apparatus further comprising a controller operable to direct the CPB source to generate CPB pulses such that each pixel of the CPB array detector is associated with a corresponding portion of each FOV in the FOV that receives an average number of less than 1 electron in the CPB pulse.

18. The CPB apparatus of claim 15, further comprising a controller operable to combine FOV images based on each FOV image bundle in the FOV image bundle to generate an FOV image.

19. The CPB apparatus of claim 14, wherein the CPB is sequentially deflected to the plurality of FOVs of the region of interest of the sample.

20. A method comprising: The electron beam is repeatedly directed to at least one field of view of the sample; as well as The at least one field of view is imaged using an array detector that defines a plurality of pixels, wherein the electron beam is configured to effectively expose the FOV region associated with the pixel to no more than a selected number of electrons.

21. The method of claim 20, wherein the electron beam is configured to effectively expose the FOV region based on direct electron beam exposure and evanescent exposure.

Images