Method for acquiring tilt-series images of sample at multiple tilt angles

Abstract

To provide a method for improving tracking of the field of view in the acquisition of tilt series images.SOLUTION: A method includes a step of acquiring a tilt series of images on the basis of exposing a region of interest of a sample to a charged particle beam at a plurality of tilt angles. The method includes a step of tracking a field of view during acquiring the tilt series of images. The method includes a step of generating a tomographic image of a sample volume associated with the region of interest on the basis of at least some of the acquired tilt series images. The step of tracking the field of view includes a step of exposing a tracking region that is substantially outside the region of interest.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method and charged particle microscope for acquiring tilt-series images based on exposure of a region of interest (ROI) of a sample to a charged particle beam (CPB) at multiple tilt angles. More specifically, the present invention relates to a system for generating detailed images of a sample utilizing a charged particle optical column, a sample holder, a charged particle detector, and an imaging system to acquire tilt-series images. [Background technology]

[0002] In a variety of scientific and industrial applications, it is often necessary to examine the microscopic structure and composition of a sample. Traditional optical microscopes have resolution limitations and are unable to provide sufficient detail for certain types of samples. To overcome these limitations, charged particle microscopes have been developed.

[0003] Charged particle microscopy is a well-known and increasingly important technique for imaging small objects, particularly in the form of electron microscopy. Historically, the basic type of electron microscope has evolved into several well-known instrument types, such as transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning transmission electron microscopes (STEMs), as well as various variants, such as so-called "dual beam" instruments (e.g., FIB-SEMs) that additionally employ a focused ion beam (FIB) to enable auxiliary activities such as ion beam milling or ion beam induced deposition (IBID). Those skilled in the art will be familiar with the different types of charged particle microscopy.

[0004] These charged particle microscopes typically include several components, which are described below.

[0005] First, a charged particle microscope includes a charged particle optical column that is used to direct a charged particle beam onto a sample, and this optical column serves to direct and control the charged particles toward the sample to ensure accurate imaging.

[0006] Additionally, charged particle microscopes include a specimen holder to hold the specimen securely in place during the imaging process. Specimen holders are typically designed to accommodate a variety of specimen sizes and shapes and to provide positioning stability and reproducibility.

[0007] Additionally, the charged particle microscope includes a charged particle detector for capturing the charged particles interacting with the sample and converting the energy or intensity of the charged particles into an electrical signal that can be further processed to generate an image.

[0008] The charged particle microscope also includes an imaging system positioned to process signals from the charged particle detector, the imaging system receiving data from the charged particle detector and generating an image signal based on the collected information.

[0009] Transmission electron microscopy (TEM) can be used to obtain high-resolution images that reveal important details of many types of samples, including biological samples. Electron beam tomography requires multiple images of a sample for image reconstruction.

[0010] Typically, tracking the field of view (FOV) in tomography is a time-consuming process. There are two main methods for tracking: "post-tracking," in which an already acquired image is used, and "pre-tracking," in which images are acquired in a nearby area before the main acquisition. However, both of these methods have drawbacks. "Post-tracking" can fail due to the low dose typically used to acquire the main image, which results in a low signal-to-noise ratio (SNR). "Pre-tracking" is more reliable because it allows for the use of a higher dose, but adds significant time to the process.

[0011] In a specific tomography method called fast incremental single exposure (FISE) acquisition, the camera is always on and never has a chance to track. This method assumes that the area of ​​interest remains within the FOV, which can be difficult or impossible due to the inevitable movement of the specimen stage during tilting. This FISE acquisition method is described in "Rapid tilt-series acquisition for electron cryotomography," Journal of Structural Biology, 2019 Feb 1, 205(2), 163-169. Summary of the Invention [Problem to be solved by the invention]

[0012] It is therefore an object of the present disclosure to provide improved methods, particularly methods for improving tracking of the field of view in tilt series acquisitions. [Means for solving the problem]

[0013] To this end, the present disclosure provides a method as set forth in claim 1. The method defined herein comprises acquiring a tilt series of images based on exposure of a region of interest (ROI) of a sample to a charged particle beam (CPB) at a plurality of tilt angles. Further, the method comprises tracking a field of view (FOV).

[0014] The field of view (FOV) tracking step is performed during the step of acquiring tilt-series images and includes exposing a tracking region that is substantially outside the region of interest (ROI). This prevents the tracking process from interfering with imaging of the ROI, reducing damage to the ROI and resulting in more accurate reconstruction of the tomographic image. Additionally, using a tracking region outside the ROI allows the FOV to be tracked during tilt-series image acquisition (especially during the actual step of tilting the sample), thereby reducing tilt-series acquisition time and improving accuracy because the FOV can be tracked during acquisition.

[0015] As defined herein, this method provides an improved method for tracking the field of view by exposing a tracking region, which is substantially distinct from the region of interest. The tracking region allows for continuous (or semi-continuous) tracking of the field of view during acquisition of tilt series images. Information obtained from this tracking region, which may be an image, can be processed to ensure that the region of interest remains within the field of view.

[0016] Thus, an improved method of acquiring tilt series is obtained, which allows for accurate tracking of the field of view during the imaging process, thereby improving over "pre-tracking" and "post-tracking," thereby achieving the objectives of the present disclosure.

[0017] Advantageous embodiments are described below.

[0018] In an embodiment, the method includes deflecting a charged particle beam between a region of interest and a tracking region. Deflecting between the two regions allows tracking of the field of view while reducing exposure to the region of interest. In this manner, dose control is applied to the region of interest.

[0019] In an embodiment, the ratio of exposure of the region of interest to exposure of the tracking region is between 1:2 and 1:10, more specifically, approximately 1:4. In other words, a ratio of 1:4 may expose the region of interest 20% of the time and the tracking region 80% of the time. Thus, during the acquisition of a tilt series, the charged particle beam is directed to the region of interest 20% of the time to collect an image of the region of interest, and the charged particle beam exposes the tracking region 80% of the time so that the tilt angle can reach the next position where the region of interest can be imaged again.

[0020] In an embodiment, the tracking region is completely separate from the region of interest, in other words there is no overlap between the tracking region and the region of interest, which further helps to protect the region of interest.

[0021] In an embodiment, the method includes determining a shift in the field of view of the region of interest using the image of the tracking region.

[0022] In an embodiment, the method includes providing a feedback loop using the field of view of the tracking region during the step of acquiring a tilt series of images of the region of interest. This feedback loop helps to continuously adjust and optimize the imaging process, improving image quality. The feedback loop allows for reliable tracking of the FOV without adding significant overhead time. The feedback loop is used in the step of tilting the sample to the next acquisition angle.

[0023] In an embodiment, the method includes using a blanker to at least partially prevent exposure to the region of interest during the step of exposing the tracking region. The (fast) blanker is used for dose control of the region of interest. A fast deflector is used to switch between the region of interest and the tracking region. The blanker can be applied as an on / off switch with a predetermined duty cycle.

[0024] In an embodiment, the method includes correcting the field of view by moving the sample relative to the charged particle beam, which allows for accurate alignment and positioning of the FOV and ensures accurate reconstruction of a tomographic image.

[0025] In embodiments, the method can be applied to both step- and sequential tilt-tomography. This versatility allows the method to be utilized with a variety of imaging techniques, providing flexibility in experimental setup.

[0026] In an embodiment, the method includes generating a tomographic image of the sample volume associated with the region of interest based on images of at least some of the acquired tilt series.

[0027] According to one aspect, there is provided a charged particle microscope as defined in claim 1. The charged particle microscope as defined herein includes a charged particle optical column for directing a charged particle beam onto a sample, a sample holder for holding the sample, a charged particle detector, and an imaging system for generating an image signal based on information from the charged particle detector. The charged particle microscope is further configured to acquire tilt series images based on exposure of a region of interest (ROI) of the sample to the charged particle beam (CPB) at a plurality of tilt angles. The charged particle microscope is further configured to track a field of view (FOV). The charged particle microscope may be configured, or may be part of a system, to generate a tomographic image of a sample volume associated with the ROI based on images of at least a portion of the acquired tilt series.

[0028] As defined herein, the charged particle microscope M is arranged to track the field of view (FOV) during the step of acquiring images of a tilt series by exposing a tracking region that is substantially outside the region of interest (ROI). The advantage of this arrangement is that it allows for the integration of tracking and imaging functions within the microscope system. Tracking the field of view using the tracking region allows tracking during the acquisition of a tilt series (i.e., during the step of tilting the sample) without significantly damaging the region of interest. Based on the data emanating from the tracking region, an optimal stage settling time based on the measured drift can be determined, and the eucentric Offsets may be detected and corrected, allowing for pre-alignment and live image analysis.

[0029] In an embodiment, the charged particle microscope is arranged to track a field of view (FOV) by exposing a tracking region that is substantially outside the region of interest (ROI), which ensures accurate tracking of the FOV without interfering with the imaging process of the ROI.

[0030] In an embodiment, the charged particle microscope is arranged to perform any of the method embodiments disclosed herein.

[0031] To provide real-time feedback to the microscope, the imaging system of the charged particle microscope may have a first charged particle detector output interface for outputting a first data stream of data associated with the charged particle detector and a second charged particle detector output interface for outputting a second data stream of data associated with the charged particle detector. These two charged particle detector output interfaces can be used to provide real-time feedback (from one output interface) while maintaining high quality and reliability of the data (from the other output interface). Having two charged particle detector output interfaces positioned to provide different data streams compared to each other allows the system to have two separate data streams that can be used for different purposes.

[0032] By way of example, the first data stream may be designed to prioritize data quality and reliability and ensure accurate and reliable data output. Thus, the first data stream may be optimized for high image quality standards that enable the generation of a tomographic image of the sample volume associated with the region of interest based on at least some images of the acquired tilt series.

[0033] The second data stream is designed to be optimized for low and / or fixed latency, allowing images of the tracked area to be acquired and used to provide real-time feedback to the charged particle system, which can be used to make relative adjustments between the sample and the charged particle beam by stage movement and / or charged particle beam deflection.

[0034] As defined herein, latency relates to the time difference between the moment a charged particle hits a charged particle detector (i.e., a charged particle camera) and the moment information from that charged particle is ready to be used by any devices located downstream of the output interface. Examples of devices that are / can be located downstream of the output interface include data storage, feedback processing devices, controllers, etc. Latency may relate to an average time difference or a maximum time difference.

[0035] In an embodiment suitable for a charged particle microscope, the latency is on the order of half the readout integration time of the charged particle detector. If the charged particle detector, which is a TEM camera, operates at 500 frames per second (fps), then each pixel takes 2 ms to be read out. Low latency means that the time it takes for data to be available from the charged particle detector and output via the second output interface (and subsequently available for use by further processing devices) is also on the order of the pixel readout. This means that the low latency needs to be on the order of ms. Based on this example, the average latency is about 1 ms, with a maximum latency of around 2 ms. Higher latency, while not optimal, can still be beneficial in providing real-time feedback.

[0036] As mentioned above, the maximum low latency output may be related to the frame rate of the charged particle detector. For fps, the maximum latency is preferably 1 / 500 = 2 ms. For lower frame rates (e.g., 250 fps), the maximum latency can be 1 / 250 = 4 ms.

[0037] As mentioned above, the average low latency output may be related to the frame rate of the charged particle detector. For a given frame rate (e.g., 250 fps), the average low latency should be around 0.5 x 1 / 500 = 1 ms. For a lower frame rate (e.g., 250 fps), the average low latency can be 0.5 x 1 / 250 = 2 ms.

[0038] The average and / or maximum latency can be used to provide real-time feedback to the charged particle microscope, allowing it to accurately track the field of view during the acquisition of a tilt series.

[0039] In an embodiment, the charged particle microscope is part of a system arranged to generate a tomographic image of a sample volume associated with a region of interest based on images of at least a portion of the acquired tilt series. [Brief explanation of the drawings]

[0040] For a more complete understanding of the present disclosure and its advantages, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Figure 1] 1 shows a longitudinal section of a charged particle microscope, in particular a transmission charged particle microscope. [Figure 2] 1 illustrates an embodiment of a charged particle microscope as defined herein. [Figure 3] 1 illustrates a further embodiment of a charged particle microscope as defined herein; [Figure 4] 1 illustrates yet another embodiment of a charged particle microscope as defined herein. [Figure 5A] 1 illustrates a region of interest and a tracking area of ​​a sample in an embodiment of the method disclosed herein. [Figure 5B] 1 illustrates a region of interest and a tracking area of ​​a sample in an embodiment of the method disclosed herein. [Figure 6A] 1 illustrates a region of interest and a tracking area of ​​a sample in an embodiment of the method disclosed herein. [Figure 6B] 1 illustrates a region of interest and a tracking area of ​​a sample in an embodiment of the method disclosed herein. [Figure 7A]10 illustrates an embodiment of a deflector signal and a blanker signal during acquisition. [Figure 7B] 10 illustrates an embodiment of a deflector signal and a blanker signal during tilting. [Figure 8] 10 illustrates an embodiment of a deflector signal and a blanker signal during tilting. DETAILED DESCRIPTION OF THE INVENTION

[0041] FIG. 1 (not to scale) is a highly schematic depiction of an embodiment of a charged particle microscope M according to an embodiment of the present invention. More specifically, it shows an embodiment of a transmission microscope M, in this case a TEM / STEM (although in the context of the present invention, an SEM (see FIG. 2) or an ion-based microscope would be equally valid). In FIG. 1, within a vacuum enclosure 2, an electron source 4 generates an electron beam B, which propagates along an electron-optical axis B′ and traverses an electron-optical illumination device 6, which serves to direct / focus the electrons onto a selected portion of a sample S (e.g., which may be (locally) thinned / flattened). Also shown is a deflector 8 that can be used to perform (among other things) a scanning movement of the beam B.

[0042] The sample S is held in a sample holder H, which can be positioned with multiple degrees of freedom, by a positioning device / stage A that moves a cradle A' to which the holder H is (removably) mounted; the sample holder H may include fingers that can move (among other things) in the XY plane (referring to the illustrated Cartesian coordinate system; typically, movement parallel to Z, and tilting about X / Y are also possible). Such movement allows different portions of the sample S to be illuminated / imaged / inspected by the electron beam B moving along axis B' (Z direction) (and / or allows a scanning movement to be performed as an alternative to beam scanning). If necessary, an optional cooling device (not shown) can be in intimate thermal contact with the sample holder H to maintain it (and the sample S thereon) at cryogenic temperatures.

[0043] The electron beam B interacts with the sample S in such a way as to emit various types of "stimulated" radiation from the sample S, including (for example) secondary electrons, backscattered electrons, X-rays, and optical radiation (cathodoluminescence). If desired, one or more of these radiation types can be detected using an analytical device 22, such as a scintillator / photomultiplier tube combination or an EDX (energy dispersive X-ray spectroscopy) module, in which case an image can be constructed using essentially the same principles as in an SEM. However, alternatively or supplementarily, electrons that traverse (pass) the sample S, are emitted, and continue to propagate (substantially, albeit with some deflection / scattering) along axis B' can be studied. Such a transmitted electron flux enters an imaging system (projection lens 24), which typically includes various electrostatic / magnetic lenses, deflectors, correctors (stigmators), etc. In normal (non-scanning) TEM mode, this imaging system 24 can focus the transmitted electron beam onto a fluorescent screen 26, which can be retracted / backed (schematically indicated by arrow 26') out of the way of axis B' as required. An image (or Fourier transform diagram) of a (portion of) the sample S is formed on the screen 26 by the projection system 24 and is viewable through a viewing port 28 located in a suitable part of the wall of the housing 2. The retraction mechanism of the screen 26 may be mechanical and / or electrical in nature and is not shown here.

[0044] Instead of viewing an image on a screen 26, one can take advantage of the fact that the depth of focus of the electron beam leaving the projection system 24 is typically very large (e.g., on the order of one meter). As a result, various other types of analytical devices can be used downstream of the screen 26, such as:

[0045] - TEM detector (camera) 30: In camera 30, the electron beam can form a still image (or Fourier transform diagram) that is processed by controller / processor 20 and displayed on a display device (not shown), such as a flat panel display. When not needed, camera 30 can be retracted / backed out of the way of axis B' (schematically indicated by arrow 30').

[0046] - STEM detector (camera) 32: The output from camera 32 can be recorded as a function of the (X,Y) scanning position of beam B on sample S, and an image can be constructed that is a "map" of the output from camera 32 as a function of X,Y. Camera 32 may be a matrix of pixels, e.g., 20 in diameter, as opposed to the matrix of pixels that is characteristically present in camera 30. Furthermore, the camera 32 generally includes a single pixel of 10 mm. 2 images / sec) than the acquisition speed (e.g., 10 6 points / sec). Again, when not needed, camera 32 can be retracted / backed out of the way of axis B' (as indicated diagrammatically by arrow 32') (such retraction is not required in the case of a donut-shaped annular dark field camera 32, although in such cameras a central hole allows the bundle to pass when the camera is not in use).

[0047] As an alternative to imaging using a camera 30 or 32, a spectroscopic detector 34 can also be invoked, which can be for example an EELS module.

[0048] It should be noted that the order / position of items 30, 32 and 34 is not strict and many possible variations are possible. For example, spectroscopic detector 34 could be integrated into imaging system 24.

[0049] In the illustrated embodiment, the microscope M includes a retractable X-ray computed tomography (CT) module generally designated by the reference numeral 40. In computed tomography (also called tomography), an electron source and a (mutually opposing) detector are used to interrogate a sample along different lines of sight so as to obtain transmitted views of the sample from various viewpoints.

[0050] It is noted that the detectors 30, 32, and 34 are part of an imaging system (generally designated by reference numeral 200). The imaging system is arranged to generate image signals based on information from the charged particle detectors 30, 32, and 34 and may be part of or separate from the detectors. A controller (computer processor) 20 is connected to the various components shown via control lines (bus) 20′. The controller 20 may provide various functions, such as synchronizing actions, providing set points, processing signals, performing calculations, and displaying messages / information on a display device (not shown). Of course, the (schematically drawn) controller 20 may be (partially) inside or outside the housing 2 and may have an integral or composite structure, as desired.

[0051] Those skilled in the art will understand that the interior of the housing 2 does not need to be maintained under strict vacuum conditions. For example, in so-called "environmental TEM / STEM," a background atmosphere of a predetermined gas is intentionally introduced / maintained within the housing 2. Those skilled in the art will also understand that in practice, it may be advantageous to limit the volume of the housing 2, if possible, to essentially enclose the axis B', taking the form of a small tube (e.g., on the order of 1 cm in diameter) through which the electron beam used passes, while expanding to accommodate components such as the electron source 4, the sample holder H, the screen 26, the camera 30, the camera 32, and the spectroscopic detector 34. Thus, the charged particle microscope M shown in FIG. 1 comprises a charged particle optical column O for directing the charged particle beam B to the sample, a sample holder H for holding the sample S, and charged particle detectors 22a, 22b, 30, 32, and 34 with an imaging system 200 for generating image signals based on information from the charged particle detectors.

[0052] 2, there is shown an embodiment of a charged particle microscope M, including further details of the charged particle detector D and imaging system 200 disclosed herein. Note that in general, detector D can be any of the TEM camera 30, STEM camera 32, spectroscopic detector 34, or segmented detector 22 shown in FIG. 1, or any other common charged particle detector suitable for acquiring tilt-series images.

[0053] 1, the charged particle microscope M includes a detector D including a detector chip 31. Raw data obtained from the detector chip 31 is transmitted to an input interface 100 of an imaging system 200. The imaging system 200 is arranged to generate an image signal based on information from the charged particle detector D.

[0054] As shown in FIG. 2, the imaging system 200 generally comprises an input interface 100 for receiving data, a first charged particle detector output interface 101 for outputting a first data stream of data associated with the charged particle detector D, and a second charged particle detector output interface 102 for outputting a second data stream of data associated with the charged particle detector D.

[0055] In Figure 2, the first charged particle detector output interface 101 is arranged to transfer data to storage 300. The connection from detector D through imaging system 200 to storage 300 is used to store high quality images from the sample. Here, the configuration of the first data stream prioritizes data quality and reliability, ensuring accurate and reliable data output to storage 300 (i.e., maintaining the high image quality standards known from current charged particle microscopes).

[0056] The imaging system also includes a second charged particle detector output interface 102. The second charged particle detector output interface 102 is arranged to provide an output different from the first output interface 101. In particular, the second charged particle detector output interface 102 is arranged to provide a low latency output to a feedback data processing device 400 connected to the microscope M. Thus, a feedback control loop is established that can be used for feedback control applications such as active image-based drift correction, continuous tilt tomography field correction, or system state measurement methods.

[0057] In the embodiment shown, the imaging system 200 comprises a data processing unit 99 arranged to process the so-called "raw" data obtained from the detector chip 31. It should be noted that this processing unit 99 may be part of the detector D itself, or the imaging system 200 may also comprise part of the detector D. Other arrangements are also possible.

[0058] After initial processing performed by processing unit 99, the signal is split into two different streams.

[0059] One stream is connected to the first charged particle detector output interface 101 and includes a storage representation module 201 and a buffer 211. The storage representation module 201 and the buffer 211 are arranged to provide high quality image data from the detector D to the storage 300 without substantially losing any image information.

[0060] The other stream is connected to the second charged particle detector output interface 102 and includes a low latency representation module 202. The low latency representation module 202 is arranged to process signals obtained from the detector D (and optionally processed by the data processing unit 99) and provide them to the second charged particle detector output interface 102. From there, they are forwarded to a feedback data processing device 400 where further processing is performed to enable real-time feedback to the charged particle microscope M. This allows low latency feedback including stage movements and / or optical changes to be provided to the charged particle microscope M.

[0061] FIG. 3 illustrates a further embodiment of a charged particle microscope M equipped with an imaging system 200 as disclosed herein. Here, the detector D includes a camera chip 31 and a data processing unit 99. The data processing unit 99 is configured to perform high-speed, "essential" processing, including gain correction and electron counting if the detector D is an electronic detector. This processing typically occurs with low latency. The resulting information is transmitted from the detector D via an output 90 to the input interface 100 of the imaging system 200. In the imaging system 200, the data is split into two different representation blocks 201 and 202. These blocks 201 and 202 are configured to convert the data into the desired output-specific representation. These blocks 201 and 202 may include additional processing steps and data expansion steps based on other information sources, such as a system clock. For example, if a user selects dose-segmented storage, the storage representation block 201 can create a dose-segmented image, and the low-latency representation block 202 can output an electronic event representation stream (a stream of electronic event positions (x, y, time)) while embedding additional timing information, such as synchronized timestamps. The storage representation 201 is buffered in block 211 so that data is not lost even if the connection to the storage 300 is briefly interrupted. The low latency representation 202 is sent without buffering (or at least without significant buffering) so as not to add extra latency.

[0062] This low latency representation module 202, in an embodiment, runs independently from the aforementioned storage representation module 201. The low latency representation module 202 is preferably optimized for low latency, while data reliability is a low priority, meaning that missing small portions of data is acceptable as long as it can be detected.

[0063] A particular type of detector D of interest is an electron counting camera, in which case the electron counting algorithm is the primary algorithm. The type of data on the output data interface 201 is either an image "movie" (called dose fractionation) or a stream of electronic event positions (x, y, time), also called an electronic event representation (EER). Providing EER data on the low-latency output data interface 202 enables low-latency event-based processing for low-dose applications. Under low-dose conditions, low-latency EER data yields the best performance for the use cases described above.

[0064] Returning to FIG. 3 , the low-latency representation module 202 is shown connected to a feedback data processing device 400, which in the illustrated embodiment is part of the controller / processor 20. Those skilled in the art will appreciate that the feedback data processing device 400 may also be a separate controller / processor. From there, the controller / processor 20 can provide feedback to the charged particle microscope M (e.g., as shown in FIG. 1 ) by changing settings / parameters for one or more of the optical column O, the sample holder H, the imaging system 24, and / or the charged particle detector D (which may be any of 22 a, 22 b, 30, 32, or 34, as shown in FIG. 1 ). In combination with the low-latency representation module 202, the feedback data processing device 400 enables real-time feedback to be provided to the charged particle microscope M.

[0065] FIG. 4 shows a schematic diagram of an embodiment of a charged particle microscope M including a controller / processor 20, which allows for the acquisition of tilt-series images as defined herein. Similar to the charged particle microscope embodiments shown in FIGS. 1-3, the charged particle microscope M shown in FIG. 4 includes a charged particle optical column O for directing a charged particle beam B to a sample S, a sample holder H for holding the sample S, and a charged particle detector D having an imaging system 200 for generating an image signal based on information from the charged particle detector. The charged particle microscope is configured to acquire tilt-series images based on exposure of a region of interest (ROI) of the sample to the charged particle beam (CPB) at multiple tilt angles. The holder H is configured to tilt the sample S to provide multiple tilt angles, as known to those skilled in the art. The charged particle microscope M is configured to track the field of view (FOV) of the region of interest. Images acquired by the microscope M can be used to generate a tomographic image of a sample volume associated with the region of interest based on at least a portion of the images in the acquired tilt series.

[0066] Detector D provides an image to controller 20, which then uses the image as feedback to microscope M to modify the holder (position, angle, etc.) to ensure the region of interest is within the field of view. Detector D can be synchronized with optical column O and / or blanker / deflector 8 that is part of optical column O. The low latency potential of detector D and imaging system 200 described herein lends itself to the feedback low shown in FIG. 4.

[0067] An embodiment of tracking of the field of view FOV of the region of interest ROI is shown in Figures 5a and 5b, where a tracking region T is used which is found to be substantially outside the region of interest ROI of the sample S.

[0068] FIG. 5a illustrates the acquisition steps for acquiring tilt series images. Tilt series images are typically acquired as a video of frames, which are then post-processed to generate a single image. Therefore, multiple frames are acquired in the region of interest, providing the opportunity to interleave these frames with tracking frames. During this tilt series acquisition, beam B may alternately expose the region of interest ROI and the tracking region T. In the illustrated embodiment, the exposure duty cycle of the region of interest ROI is 20%, and the exposure duty cycle of the tracking region is 80%. In other words, the region of interest is exposed approximately 20% of the time, and the tracking region is exposed the remaining 80% of the time. 20% and 80% may refer to the total number of frames acquired during acquisition.

[0069] The above figures are an example of how to distribute the dose between exposure and tracking. As shown in Figure 5a, you can use 20% of the frame for exposure and 80% of the frame for tracking ("slow mode"), or you can use 20% of the frame time of even frames for exposure and 80% of the frame time of odd frames for tracking ("fast mode").

[0070] In the so-called slow mode, one frame can be acquired during exposure and four frames during tracking, ie the total period is (1+4=) 5 frames.

[0071] In fast mode, it is possible to expose 20% of the first frame and track 80% of the second frame. Thus, switching occurs frame by frame. At exactly the same time, for example during a tilt, one can switch to "no exposure" by simply blanking the beam when there is an exposure (Figure 5b). The same can be achieved with a deflector, but it is more practical to keep the deflector signal constant and activate the (pre-programmed) blanker signal.

[0072] In an embodiment (not shown), it is possible to use the "fast mode" as described above, slightly modified using a blanker as follows: Here, the blanker can be used to unblank the charged particle beam for only a short time window within a frame when no frame readout is occurring. The detector may be positioned to add a short "no readout" period to each frame time, slightly slowing the frame rate and allowing for a "clean" exposure. Without this, some lines would be exposed but still count towards the "old" frame, causing the frames to "blur into each other over time."

[0073] Figure 5b shows a step in which (after the acquisition of the tilt angle according to Figure 5a) the tilt angle is changed to a new angle (i.e., no tilt series images are acquired, but the microscope is set to acquire new tilt series images). Here, the tracking region T is still exposed to the charged particle beam, but the region of interest ROI is not exposed at all. The region of interest ROI can be prevented from being exposed by blanking and / or deflecting the charged particle beam B. For this purpose, a deflector or blanker 8 known to those skilled in the art can be used.

[0074] Thus, in Figures 5a and 5b, during the acquisition of a tilt series image and during preparation of the next tilt series image, exposure of the tracking region T is performed while manipulating the stage H to move to a different stage angle. This allows the tracking region to be used to track the field of view FOV and align the region of interest with the beam B of the charged particle microscope M. Thus, the field of view can be aligned and corrected during the acquisition of a tilt series.

[0075] Figures 6a and 6b show an alternative embodiment, differing primarily in how the beam is used to expose the region of interest ROI for tilt series image acquisition. Here, the region of interest ROI is exposed to beam B during tilt series image acquisition. While the region of interest ROI is exposed, the tracking region is not exposed and is not exposed by the charged particle beam. After fully exposing the region of interest ROI, the beam is shifted a predetermined amount (using deflector 8) to ensure that the beam exposes tracking region T. The tracking region is then tracked while moving to the next tilt angle during stage H manipulation (Figure 6b). This allows tracking region T to be used to track the field of view (FOV) of the region of interest ROI, allowing stage H and / or beam B to be modified to maintain the field of view at the correct region of interest.

[0076] In Figures 6a and 6b, the region of interest ROI and the tracking region T are alternately exposed: the region of interest ROI during tilt series image acquisition, and the tracking region T during the tilt. This is, in principle, the simplest version and can be realized without the need for a high-speed deflector or blanker.

[0077] In principle, there are three scenarios for establishing the exposure of the region of interest ROI while tracking the tracking region T.

[0078] Scenario A (Figures 7a and 7b): The simplest way to achieve the situation in the example of Figure 5 is to synchronize the blanker 8 with the camera D and distribute the frames 1:4 between the exposure and tracking regions. The deflector and blanker signals are shown in Figures 7a and 7b. Figure 7a shows the deflector signal 701 and blanker signal 702 during acquisition, while Figure 7b shows the deflector signal 701 and blanker signal 702 during tilt. The beam B simply switches between the area ROI and T, and is blanked in the region of interest ROI during tilt. This is the situation shown in Figures 6a and 6b.

[0079] Scenario B (Figure 8): To allow faster acquisition, the beam can instead be switched every frame, reducing the duty cycle in the exposure area to 20% using blanker 8. Acquire faster with the same duty cycle (20% exposure, 80% tracking). During the tilt, the blanker switches from 20% to 0% duty cycle in the exposure area, see deflector signal 801 and blanker signal 802 in Figure 8).

[0080] Scenario C: During acquisition, the beam is kept in the exposure area. This allows using every camera frame (instead of every second frame) during the exposure, improving the temporal resolution. This does not require constantly switching the deflector signal (not shown) so that both the region of interest ROI and the tracking region T are imaged simultaneously.

[0081] Although the duty cycles shown in Figures 6-8 and described in Scenarios A, B, and C are shown to sum to 100%, this is not necessary. For example, in a region of interest (ROI), only a 1% duty cycle may be used (i.e., stroboscopic illumination).

[0082] Furthermore, note that scenarios A and B can be combined by changing five independent parameters: (1) the length of the slow (frame-wide) period; (2) slow duty cycle of the region of interest ROI; (3) the slow duty cycle of the tracking region T; (4) Fast (within frame) duty cycle of the region of interest (ROI); (5) Fast (intraframe) duty cycle of the tracking region.

[0083] From the above description, it will be understood that a charged particle microscope M is positioned, and the method described herein includes tracking a field of view (FOV) during a step of acquiring tilt series images by exposing a tracking region substantially outside the region of interest (ROI). Acquiring tilt series images includes an exposure step of exposing the region of interest to acquire tilt series images, and a tilt step of tilting the sample to a new tilt angle. Thus, it will be understood that "during the step of acquiring tilt series images" may include exposing the tracking region T between successive exposures of the region of interest ROI at two different tilt angles. It will further be understood that "during the step of acquiring tilt series images" may also include exposing the tracking region T during a tilt step of tilting the sample to a new tilt angle.

[0084] The methods and charged particle microscope M described herein allow reliable tracking of the field of view FOV without overhead.

[0085] This technique can be used for tilt tomography and continuous tilt tomography.

Claims

1. acquiring a tilt series of images based on exposure of a region of interest of the specimen to a charged particle beam at a plurality of tilt angles; and tracking a field of view of the region of interest, 10. The method of claim 9, wherein the step of tracking the field of view is performed during the step of acquiring the tilt series of images and includes exposing a tracking region that is substantially outside the region of interest.

2. The method of claim 1 , comprising deflecting the charged particle beam between the region of interest and the tracking region.

3. The method of claim 2 , wherein the ratio of exposure of the region of interest to exposure of the tracking region is between 1:2 and 1:10, more particularly about 1:

4.

4. The method of claim 1 , wherein the tracking region is completely separate from the region of interest.

5. The method of claim 1 , comprising determining a shift in field of view of the region of interest using images of the tracking region.

6. The method of claim 1 , comprising using the field of view of the tracking region to provide a feedback loop during the step of acquiring tilt-series images of the region of interest.

7. The method of claim 1 , further comprising using a blanker to at least partially prevent exposure to the region of interest during the step of exposing the tracking region.

8. The method of claim 1 , comprising correcting the field of view by moving the sample relative to the charged particle beam.

9. The method of claim 1 , wherein the method comprises step-based tomography or sequential tilt tomography.

10. a charged particle optical column for directing the charged particle beam onto the sample; a sample holder for holding a sample; a charged particle detector; an imaging system for generating an image signal based on information from the charged particle detector, The charged particle microscope comprises: acquiring a tilt series of images based on exposure of a region of interest of a specimen to the charged particle beam at a plurality of tilt angles; tracking a field of view of the region of interest; 10. The charged particle microscope, characterized in that the charged particle microscope is configured to track the field of view during the step of acquiring the tilt series of images by exposing a tracking region that is substantially outside the region of interest.

11. 11. The charged particle microscope according to claim 10, A charged particle microscope configured to carry out the method according to any one of claims 1 to 9.

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