Method for obtaining sample tilt series images at multiple tilt angles

Abstract

A method and charged particle microscope for obtaining a tilted series of images based on exposing a region of interest of a sample to a charged particle beam at multiple tilt angles. The method comprises the step of tracking a field of view (FOV) during the step of obtaining the tilted series of images. The method comprises the step of generating a sample volume tomography image related to the region of interest based on at least a portion of the image in the obtained tilted series. As defined herein, the step of tracking the field of view (FOV) includes the step of exposing a tracking area substantially outside of the region of interest (ROI). Charged particle microscopes as defined herein are arranged for performing the methods as defined herein.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method and a charged particle microscope for obtaining a tilt series of images based on exposing 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 that generates detailed sample images for acquiring said tilt series of images using a charged particle optical column, a sample holder, a charged particle detector and an imaging system. BACKGROUND

[0002] In various scientific and industrial applications, it is often necessary to examine the microstructure and composition of a sample. Conventional optical microscopes have limitations in terms of resolution and cannot provide sufficient detail for certain types of samples. To overcome the limitations, charged particle microscopes have been developed.

[0003] Charged particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy. Historically, the basic categories of electron microscopes have evolved into a number of well-known device classes, such as transmission electron microscopes (TEM), scanning electron microscopes (SEM) and scanning transmission electron microscopes (STEM), while various sub-classes have also evolved, such as so-called "dual beam" devices (e.g. FIB-SEM) that additionally employ a focused ion beam (FIB) to allow supporting activities such as ion beam milling or ion beam induced deposition (IBID). The skilled person will be familiar with the different categories of charged particle microscopes.

[0004] The charged particle microscope typically comprises several components, which will be explained below.

[0005] Firstly, the charged particle microscope comprises a charged particle optical column with which a charged particle beam is directed onto a sample. The optical column is responsible for directing and controlling the charged particles to the sample, ensuring accurate imaging.

[0006] Furthermore, the charged particle microscope comprises a sample holder for securely fixing the sample in place during the imaging process. The sample holder is typically designed to accommodate various sample sizes and shapes, ensuring positioning stability and reproducibility.

[0007] In addition, the charged particle microscope comprises a charged particle detector for capturing the charged particles that have interacted with the sample. The charged particle detector converts the energy or intensity of the charged particles into an electrical signal, which can be further processed for image generation.

[0008] The charged particle microscope further comprises an imaging system arranged for processing signals from the charged particle detector. The imaging system receives data from the charged particle detector and generates an image signal based on the collected information.

[0009] Transmission electron microscopes (TEM) can be used to obtain high resolution images revealing important details of a variety of samples, including biological samples. In electron tomography, image reconstruction requires multiple images of the sample.

[0010] In tomography, the process of tracking the field of view (FOV) is often very time consuming. There are mainly two tracking methods: “post tracking” (where images already captured are used) and “pre tracking” (images captured at nearby regions before the main acquisition is performed). But both methods have their drawbacks. “Post tracking” can fail due to the low signal-to-noise ratio (SNR) caused by the low dose usually used for acquiring the main images. “Pre tracking” can use a higher dose and thus be more reliable, but greatly prolongs the process time.

[0011] In a specific tomography method, the so-called fast incremental single exposure (FISE) acquisition, the camera is always on and there is no opportunity to perform tracking at all. The method assumes that the field of view preserves the region of interest, which can be very difficult or even unachievable due to the inevitable movement of the sample stage when tilted. Fast incremental single exposure acquisition is described in detail in “Fast tilt series acquisition for electron cryotomography. Journal of Structural Biology Volume 205, Issue 2, 1 February 2019, Pages 163-169”. SUMMARY

[0012] Therefore, the disclosure aims to provide an improved method, in particular an improved method for tracking the field of view in a tilt series acquisition.

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

[0014] The step of tracking the field of view (FOV) is performed during the step of obtaining the tilt series images and comprises exposing a tracking region substantially outside the region of interest (ROI). This step can ensure that the tracking process does not interfere with the imaging of the region of interest, thereby reducing the damage to the region of interest and enabling a more accurate tomographic image reconstruction. Furthermore, by using a tracking region outside the region of interest, the field of view can be tracked during the acquisition of the tilt series images, in particular during the actual step of tilting the sample, thereby speeding up the acquisition for the tilt series while improving the accuracy of the ability to track the field of view during the acquisition.

[0015] As defined herein, the method provides a means of improving the tracking field of view by exposing a tracking region. The tracking region is significantly different from the region of interest. The tracking region allows for continuous (or semi-continuous) tracking of the field of view during acquisition of a tilt series of images. Information from the tracking region (e.g., which may be an image) can be processed and used to ensure that the region of interest remains within the field of view.

[0016] This results in an improved method for acquiring tilt series that accurately tracks the field of view during the imaging process, providing both "before tracking" and "after tracking" improvements. The objectives of this disclosure are thereby achieved.

[0017] Advantageous embodiments are discussed below.

[0018] In an embodiment, the method includes the steps of: deflecting the charged particle beam between the region of interest and the tracking region. By deflecting between the two regions, a field of view can be tracked while reducing exposure to the region of interest. The dose control method 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, and more specifically, about 1:4. In other words, at a ratio of 1:4, the region of interest may be exposed 20% of the time, while the tracking region may be exposed 80% of the time. Thus, during acquisition of the 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 80% of the time to expose the tracking region so that the tilt angle can be adjusted again to reach the next position where the region of interest can be imaged.

[0020] In an embodiment, the tracking area is completely separated from the region of interest. In other words, there is no overlap between the tracking area and the region of interest. The method further helps to protect the region of interest.

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

[0022] In an embodiment, the method involves using a tracking region field of view to provide a feedback loop during the step of obtaining a series of tilt images of a region of interest. The feedback loop facilitates continuous adjustment and optimization of the imaging process, thereby improving image quality. The feedback loop allows for reliable tracking of the field of view without significantly increasing operating time. The feedback loop can be used during the step of tilting the sample to the next acquisition angle.

[0023] In embodiments, the method comprises using a shutter during the step of exposing the tracking area, at least partially preventing exposure of the region of interest. A (fast) blocker can be used for dose control of the region of interest. A fast deflector can be used to switch between the region of interest and the tracking area. For example, the blocker can be used as an on / off switch with a predetermined duty cycle.

[0024] In embodiments, the method involves correcting the field of view by moving the sample relative to the charged particle beam. The correction allows for accurate alignment and positioning of the field of view, ensuring accurate tomographic image reconstruction.

[0025] In embodiments, the method can be applied to step-and-tomography and continuous tilt tomography. This versatility allows the method to be used in various imaging techniques, providing flexibility for experimental setups.

[0026] In embodiments, the method comprises generating a sample volume tomographic image related to the region of interest based on at least part of the images of the tilt series.

[0027] According to an aspect, there is provided a charged particle microscope as defined in claim 1. The charged particle microscope as defined herein comprises a charged particle optical column for directing a charged particle beam onto a sample, a sample holder for holding a sample, and a charged particle detector and imaging system for generating an image signal based on information from the charged particle detector. Further, the charged particle microscope is arranged for obtaining a tilt series of images based on exposing a region of interest (ROI) of the sample to a charged particle beam (CPB) at a plurality of tilt angles. Further, the charged particle microscope is arranged for tracking a field of view (FOV). The charged particle microscope can be arranged to generate a sample volume tomographic image related to the region of interest based on at least part of the images of the tilt series, or to generate a part of the tomographic image system.

[0028] As defined herein, the charged particle microscope M is arranged for tracking a field of view (FOV) during the step of obtaining a tilt series of images by exposing a tracking area substantially outside the region of interest (ROI). This arrangement has the advantage of allowing integration of tracking and imaging functionality within the microscope system. Tracking the field of view using a tracking area allows tracking without significant damage to the region of interest during acquisition of the tilt series, i.e. during the step of tilting the sample. Based on data emitted from the tracking area, optimal stage stabilization times based on measured drifts can be determined, and center shifts can be detected and corrected, allowing for pre-alignment and real-time image analysis.

[0029] In embodiments, the charged particle microscope is arranged for tracking a field of view (FOV) by exposing a tracking area substantially outside a region of interest (ROI). This arrangement can ensure accurate tracking of the field of view without interfering with the imaging process of the region of interest.

[0030] In embodiments, the charged particle microscope is arranged for performing any of the embodiments of the methods as disclosed herein.

[0031] To provide real-time feedback to the microscope, the imaging system of the charged particle microscope can have a first charged particle detector output interface for outputting a first data stream of data related to the charged particle detector and a second charged particle detector output interface for outputting a second data stream of data related to the charged particle detector. The two charged particle detector output interfaces can be used to provide real-time feedback (from one output interface) while maintaining a higher data quality and reliability (from the other output interface). By having two charged particle detector output interfaces, both interfaces are arranged for providing different data streams compared to each other, the system allows having two independent data streams, which can be used for different purposes.

[0032] For example, the first data stream can be designed to prioritize data quality and reliability, ensuring accurate and reliable data output. Thus, the first data stream can be optimized for high image quality standards, which allow for generating a sample volume tomographic image related to the region of interest based on at least part of the obtained tilt series.

[0033] The second data stream can be designed to be optimized for low latency and / or fixed latency. It allows obtaining images of the tracking area and using the images for providing real-time feedback to the charged particle system. The real-time feedback can be used to provide relative adjustments between the sample and the charged particle beam, for example by stage movement and / or deflection of the charged particle beam.

[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 device located downstream of the output interface. Said devices located downstream of the output interface are exemplified by data storage devices, feedback processing devices, controllers, etc. Latency can relate to an average time difference or a maximum time difference.

[0035] In embodiments suitable for charged particle microscopes, the latency is about half the readout integration time of the charged particle detector. If the charged particle detector (which can be a TEM camera, for example) runs at 500 frames per second (fps), then the pixel readout takes 2 ms. The low latency means that the time required for the data to come from the charged particle detector and be output through the second output interface (which can then be used by further processing means, for example) is also on the order of the pixel readout. This means that the low latency should be on the order of ms. Based on this example, the average latency can be about 1 ms and the maximum latency can be about 2 ms. Higher latencies can still have a beneficial impact on providing real-time feedback, even if they are not optimal.

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

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

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

[0039] In embodiments, the charged particle microscope is part of a tomography imaging system arranged for generating a tomographic image of a sample volume related to a region of interest based on at least part of the obtained tilt series. BRIEF DESCRIPTION OF DRAWINGS

[0040] For a more complete understanding of the present disclosure and its advantages, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0041] Figure 1 A longitudinal cross-sectional view of a charged particle microscope, in particular a transmission charged particle microscope, is shown;

[0042] Figure 2 An embodiment of a charged particle microscope as defined herein is shown;

[0043] Figure 3 Another embodiment of a charged particle microscope as defined herein is shown;

[0044] Figure 4 Yet another embodiment of a charged particle microscope as defined herein is shown;

[0045] Figure 5a and Figure 5b shows a region of interest and a tracking region of a sample in an embodiment of a method as disclosed herein;

[0046] Figure 6a and Figure 6b shows a region of interest and a tracking region of a sample in an embodiment of a method as disclosed herein;

[0047] Figure 7a and Figure 7b Implementations of the deflector and blocker signals during acquisition and during tilt are shown, respectively.

[0048] Figure 8 An embodiment of the deflector and blocker signals during tilt is shown. DETAILED DESCRIPTION

[0049] Figure 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 particularly, an embodiment of a transmission type microscope M is shown, which in this case is a TEM / STEM (although, for example, in the context of the present invention, it may be effectively just a SEM (see Figure 2 ) or ion-based microscopy). Figure 1 As shown, within a vacuum housing 2, an electron source 4 generates an electron beam B that propagates along an electron-optical axis B' and passes through an electron-optical illuminator 6 for directing / focusing the electrons onto a selected portion of a sample S (which may, for example, be (locally) thinned / planarized). A deflector 8 is also depicted, which can be used (among other things) to achieve a scanning motion of the beam B.

[0050] The sample S is held on a multi-degree-of-freedom positionable sample holder H by a positioning device / stage A that moves a bracket A' to which the holder H is (removably) attached; for example, the sample holder H may include fingers that can be moved (especially) in the XY plane (see Cartesian coordinate system; movement parallel to Z is generally possible, and tilting about X / Y is also possible). This movement allows different parts of the sample S to be illuminated / imaged / inspected by an electron beam B traveling along the B' axis (in the Z direction) (and / or allows scanning motion to be performed as an alternative to beam scanning). If desired, an optional cooling device (not depicted) can be brought into close thermal contact with the sample holder H, for example to maintain the sample holder (and the sample S thereon) at a low temperature.

[0051] The electron beam B will interact with the sample S in the manner described, such that various types of "stimulated" radiation are emitted from the sample S, including, for example, secondary electrons, backscattered electrons, X-rays, and optical radiation (cathodoluminescence). If desired, one or more of these types of radiation can be detected by means of an analysis device 22, which can be a combination scintillator / photomultiplier tube or an Energy-Dispersive X-Ray Spectroscopy (EDX) module, etc.; in this case, an image can be constructed using essentially the same principles as in a SEM. However, other means can also be chosen or supplemented, and electrons that pass through (by) the sample S, exit / emit from the sample and continue to propagate along the axis B' (essentially so, but often with some deflection / scattering) can be investigated. This transmitted electron flux enters a projection system (projection lens 24), which typically includes various electrostatic / magnetic lenses, deflectors, correctors (such as stigmators), etc. In normal (non-scanning) TEM mode, the projection system 24 can focus the transmitted electron flux onto a phosphor screen 26, which can be retracted / withdrawn (as indicated schematically by arrow 26') if desired, so as to be taken out of the axis B'. An image (or diffraction pattern) of the sample S (part) will be formed by the projection system 24 onto the screen 26, and can be viewed through a viewing port 28 in a suitable portion of the outer wall of the housing 2. The retraction mechanism of the screen 26 can be mechanical and / or electric, for example, and is not described herein.

[0052] As an alternative to viewing the image on the screen 26, the fact that the focal depth of the electron flux exiting the projection system 24 is typically quite large (e.g., on the order of 1 meter) can be exploited. Thus, various other types of analysis devices can be used downstream of the screen 26, such as:

[0053] - a TEM detector (camera) 30. For example, at the camera 30, the electron flux can form a static image (or diffraction pattern) that can be processed by the controller / processor 20 and displayed on a display device (not depicted) (flat panel display, etc.). The camera 30 can be retracted / withdrawn (as indicated schematically by arrow 30') if not needed, so as to be taken out of the axis B'.

[0054] - a STEM detector (camera) 32. The output from the camera 32 can be recorded as a function of (X,Y) scan position of the beam B on the sample S, and an image can be constructed from the "map" of camera 32 output as a function of X,Y. The camera 32 can comprise a single pixel, for example, 20 mm in diameter, rather than a matrix of pixels as is characteristic of the camera 30. Furthermore, the camera 32 will typically have a much higher acquisition rate (e.g., 10 2 images per second) than the camera 30 (e.g., 10 6Likewise, when not needed, the camera 32 can be retracted / withdrawn (as schematically indicated by arrow 32'), so as to avoid the axis B' (but in the case of a donut-shaped dark- field camera 32, such retraction is not necessary; in such a camera, the central hole will allow flux through when the camera is not in use).

[0055] - As an alternative to imaging using the camera 30 or 32, also a spectrometer detector 34 can be invoked, which can for instance be an EELS module.

[0056] It is noted that the order / position of items 30, 32 and 34 is not strict, and many possible variations can be envisaged. For instance, the spectrometer detector 34 can also be integrated into the projection system 24.

[0057] In the illustrated embodiment, the microscope M further comprises a scalable X-ray computed tomography (CT) module, indicated as a whole by reference numeral 40. In computed tomography (also referred to as tomographic imaging), the source and (directly opposite) detector are used to view the sample along different lines of sight, so as to obtain a penetrating view of the sample from various perspectives.

[0058] It is noted that the detectors 30, 32, 34 are part of an imaging system, indicated as a whole by reference numeral 200. The imaging system is arranged for generating an image signal based on information from the charged-particle detector 30, 32, 34, and can be part of the detector or separate therefrom. A controller (computer processor) 20 is connected to the various illustrated components via control lines (buses) 20'. The controller 20 can provide a variety of functions, such as synchronizing actions, providing set points, processing signals, performing calculations, and displaying messages / information on a display device (not depicted). Not to mention, the controller 20 (schematically depicted) can be located (partly) inside or outside the housing 2, and can have an integral or composite structure as desired.

[0059] The skilled person will understand that the interior of the housing 2 need not be kept under strict vacuum; for instance, in so-called "ambient TEM / STEM", a background atmosphere of a given gas is intentionally introduced / maintained within the housing 2. The skilled person will also understand that in practice, it can be advantageous to limit the volume of the housing 2, so as to substantially enclose the axis B in case, in the form of a small tube (e.g. of about 1 cm diameter), through which the employed electron beam passes, but which can be widened to accommodate structures such as the source 4, the sample holder H, the screen 26, the camera 30, the camera 32, the spectrometer detector 34, etc.

[0060] Thus, Figure 1The illustrated charged particle microscope M comprises a charged particle optical column O for directing a charged particle beam B onto a sample, a sample holder H for holding a sample S, and a charged particle detector 22a, 22b, 30, 32, 34 with an imaging system 200 for generating an image signal based on information from the charged particle detector.

[0061] Turning now to Figure 2 , Figure 2 An embodiment of a charged particle microscope M is shown, including more details of a charged particle detector D and an imaging system 200 as disclosed herein. It should be noted that the detector D can generally be any of a TEM camera 30, a STEM camera 32, a spectroscopic detector 34 or a segmented detector 22 (as shown in Figure 1 ), or generally any other charged particle detector, as long as the detector is suitable for acquiring a tilt series of images.

[0062] Similarly to Figure 1 , the charged particle microscope M comprises a detector D, which comprises a detector chip 31. Raw data from this detector chip 31 is sent 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.

[0063] As shown in Figure 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 related to the charged particle detector D, and a second charged particle detector output interface 102 for outputting a second data stream of data related to the charged particle detector D.

[0064] In Figure 2 , the first charged particle detector output interface 10 is arranged for transferring data to a storage device 300. The connection from the detector D via the imaging system 200 to the storage device 300 is used for storing high quality images from the sample. The first data stream is set such that data quality and reliability are prioritized, thereby ensuring accurate and reliable data output to the storage device 300 (i.e. maintaining the high image quality standards known from current charged particle microscopes).

[0065] The imaging system further comprises a second charged particle detector output interface 102. The second charged particle detector output interface 102 is arranged for providing a different output compared to the first output interface 101. In particular, the second charged particle detector output interface 102 is arranged for providing a low latency output to a feedback data processing device 400, which in turn is connected to the microscope M. Thereby a feedback control loop is established, which can be used for feedback control applications such as image based active drift compensation, continuous tilt tomography field correction or system state measurement methods.

[0066] In the shown embodiment, the imaging system 200 comprises a data processing unit 99, which is arranged for processing so-called "raw" data from the detector chip 31. It should be noted that the processing unit 99 can be part of the detector D itself, so that the imaging system 200 can also comprise part of the detector D. Other arrangements are also conceivable.

[0067] After the initial processing by the processing unit 99, the signal can be split into two different streams.

[0068] One stream is connected to the first charged particle detector output interface 101, comprising a storage representation module 201 and a buffer 211. The storage representation module 201 and the buffer 211 are arranged for providing high quality image data from the detector D to a storage device 300, substantially without any loss of image information.

[0069] The other stream is connected to the second charged particle detector output interface 102, comprising a low latency representation module 202. The low latency representation module 202 is arranged to process the signal from the detector D (and can optionally be processed by the data processing unit 99) and to provide the signal to the second charged particle detector output interface 102. From there it can be forwarded to the feedback data processing device 400, where additional processing can be performed for enabling real-time feedback to the charged particle microscope M. Thereby low latency feedback can be provided to the charged particle microscope M, which can include stage movement and / or optical changes.

[0070] Figure 3Another embodiment of a charged particle microscope M with an imaging system 200 as disclosed herein is shown. In this embodiment, the detector D includes a camera chip 31 and a data processing unit 99. The data processing unit 99 is arranged to perform fast "basic" processing, which may include gain correction and electron counting in the case where the detector D is an electron detector. The processing is generally performed with low latency. The resulting information is sent from the detector D via output 90 toward the input interface 100 of the imaging system 200. In the imaging system 200, the data is divided into two different representation blocks 201 and 202. 201 and 202 can be arranged to convert the data into a desired output-specific representation. The representation blocks 201 and 202 can include additional processing steps and data enhancement steps based on other information sources such as the system clock. For example, if the user selects dose fraction storage, the storage representation block 201 will create a dose fraction image, while the low-latency representation block 202 can output a stream of electron event representations (a stream of electron event positions (x, y, time)) while embedding additional timing information such as synchronization timestamps. The storage representation 201 is buffered in the representation block 211 to ensure that no data is lost in the event of a brief interruption in the connection to the storage device 300. The low latency representation 202 is transmitted without buffering (or at least without a large buffer) to prevent adding additional delay.

[0071] In an embodiment, the low-latency representation module 202 can run independently of the aforementioned storage representation module 201. The low-latency representation module 202 is optimized for low latency first, while data reliability has a lower priority: this means that a small amount of data loss is allowed as long as it can be detected.

[0072] One particular type of detector D of interest is an electron-counting camera: in this case, the electron-counting algorithm is the key algorithm. The data type on output data interface 201 can be an image "movie" (referred to as a dose fraction) or a stream of electron event positions (x, y, time), also known as an electron event representation (EER). Providing EER data on a low-latency output data interface 202 allows for low-latency event-based processing for low-dose applications. Under low-dose conditions, low-latency EER data will yield the best possible performance in the aforementioned use cases.

[0073] Back to Figure 3 , shows that the low-latency representation module 202 can be connected to a feedback data processing device 400, which in the illustrated embodiment is part of the controller / processor 20. It will be clear to those skilled in the art that the feedback data processing device 400 can also be a separate controller / processor. The controller / processor 20 can thereby provide feedback to the charged particle microscope M (e.g., Figure 1The method is to set / alter parameters to one or more of the optical column O, the sample holder H, the imaging system 24 and / or the charged particle detector D (which can be as Figure 1 The feedback data processing apparatus 400 and the low latency representation module 202 together allow real-time feedback to be provided to the charged particle microscope M.

[0074] Figure 4 A schematic of an embodiment of a charged particle microscope M comprising a controller / processor 20 is shown, with which a tilt series of images as defined herein can be obtained. As with the embodiment of a charged particle microscope as Figure 1 to Figure 3 As with the embodiment of a charged particle microscope as Figure 4 The charged particle microscope M as shown comprises a charged particle optical column O for directing a charged particle beam B onto a sample S, a sample holder H for holding the sample S, and a charged particle detector D with an imaging system 200 for generating an image signal based on information from the charged particle detector. The charged particle microscope is arranged for obtaining a tilt series of images based on exposing a region of interest (ROI) of the sample to the charged particle beam (CPB) at a plurality of tilt angles. As is known to the skilled person, the holder H is arranged for tilting the sample S to provide the plurality of tilt angles. The charged particle microscope M is arranged for tracking a field of view (FOV) of the region of interest. The images obtained with the microscope M can be used to produce a tomographic image of a sample volume related to the region of interest based on at least part of the obtained tilt series of images.

[0075] The detector D provides an image to the controller 20, which can then be used as feedback to the microscope M to modify the holder (position, angle, etc.) to ensure that the region of interest is within the field of view. The detector D can be synchronized with the optical column O and / or the blocker / deflector 8 as part of the optical column O. The low latency possibility of the detector D and the imaging system 200 as described herein facilitates Figure 4 The feedback as shown is low.

[0076] Embodiments of tracking a region of interest (ROI) field of view (FOV) are shown in Figure 5a and Figure 5b Here the tracking region is used, it can be seen that the tracking region T is substantially outside the region of interest (ROI) of the sample S.

[0077] Figure 5aThe acquisition steps for acquiring a tilt series of images are shown. The tilt series of images is typically acquired in video frames, which are subsequently post-processed to yield individual images. Thereby multiple frames are acquired at the region of interest, giving the opportunity to interleave the frames with tracking frames. During the tilt series acquisition the beam B can alternate exposing a region of interest (ROI) and a tracking region T. In the shown embodiment the exposure of the region of interest (ROI) has a duty cycle of 20% and the exposure of the tracking region has a duty cycle of 80%. In other words, the region of interest is exposed for about 20% of the time and the tracking region is exposed for the remaining 80% of the time. The 20% and 80% can refer to the total frames acquired during the acquisition.

[0078] The above numbers are an example of how the dose is split between exposure and tracking. Instead of using 20% of the frames for exposure and 80% of the frames for tracking ("slow mode"), it is also possible to use 20% of the frame time in even frames for exposure and 80% of the frame time in odd frames for tracking ("fast mode"), as shown in Figure 5a

[0079] In the so-called slow mode, one frame can be acquired when exposing and four frames when tracking, i.e. a total cycle of (one plus four =) five frames.

[0080] In the fast mode, 20% of the first frame can be used for exposure and 80% of the second frame for tracking. The switching occurs once per frame. Using exactly the same timing, e.g. by simply blanking the beam Figure 5b ) when there is exposure, it is possible to switch to "no exposure". The same can be achieved using a deflector, but it is more practical to keep the deflector signal unchanged and only activate the (pre-programmed) blanking signal.

[0081] In an embodiment (not shown) the above "fast mode" can be used and then the blocker is slightly modified. When there is no frame readout, the blocker can be used to eliminate the charged particle beam for only a short time window within the frame. The detector can be arranged to add a short "no readout" time to each frame time. The frame rate can be slightly reduced, achieving a "clean" exposure. If this is not done, part of the line will be exposed, but still counted into the "old" frame, resulting in frames "blurring into each other in time".

[0082] Figure 5b The steps are shown, wherein (in accordance with the above) Figure 5a ​The tilt angle is changed to a new angle (i.e. no tilt series of images is being acquired, but the microscope is being set up to acquire a new tilt series of images). The tracking area T is still exposed to the charged particle beam, while the region of interest (ROI) is completely unexposed. The region of interest (ROI) can be left unexposed by blocking and / or deflecting the charged particle beam B. For this purpose, a deflector or blocker 8 known to the person skilled in the art can be used.

[0083] Thus, in Figure 5a and Figure 5b , the exposure of the tracking area T is performed during the acquisition of the tilt series of images and during the preparation of the next tilt series of images, i.e. during the manipulation of the stage H to move to another stage angle. The tracking area is allowed 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 the tilt series.

[0084] Figure 6a and Figure 6b shows an alternative embodiment, the main difference being in the way the beam is used during the exposure of the region of interest (ROI) to acquire the tilt series of images. During the acquisition of the tilt series of images, the region of interest (ROI) is exposed to the beam B. While the region of interest ROI is exposed, the tracking area is not exposed, i.e. it is shielded from the charged particle beam. After the region of interest (ROI) is sufficiently exposed, the beam is moved by a predetermined amount (using the deflector 8) ensuring that the beam exposes the tracking area T. The tracking area (T) is then tracked during the manipulation of the stage H during the movement to the next tilt angle. This allows to track the field of view (FOV) of the region of interest (ROI) using the tracking area T and allows to modify the stage H and / or the beam B to keep the field of view in the correct region of interest. Figure 6b ) during the manipulation of the stage H. This allows to track the field of view (FOV) of the region of interest (ROI) using the tracking area T and allows to modify the stage H and / or the beam B to keep the field of view in the correct region of interest.

[0085] In Figure 6a and Figure 6b , the region of interest (ROI) and the tracking area T are alternately exposed. The region of interest (ROI) during the acquisition of the tilt series of images and the tracking area T during the tilting. In principle, this is the simplest version, which can be achieved without a fast deflector or blocker.

[0086] In principle, there are three scenarios for establishing the exposure of the region of interest (ROI) while keeping track of the tracking area T.

[0087] Scenario A Figure 7a and Figure 7b : The simplest way to implement the case as in the example in Fig. 5 is to synchronize the blocker 8 with the camera D and to distribute the frames between the exposure and the tracking area by 1 :4. The deflector and blocker signals are as Figure 7a andFigure 7b are shown. Figure 7a The deflector 701 and blocker 702 signals during acquisition are shown, Figure 7b The deflector 701 and blocker 702 signals during tilt are shown. The beam B is just switched between the regions of interest, T regions, and is blocked at the region of interest (ROI) during tilt. This is as Figure 6a and Figure 6b shown.

[0088] Scenario B Figure 8 ): To speed up acquisition, the beam can be switched once per frame instead, and the duty cycle at the exposure region reduced to 20% using the blocker 8. The acquisition is faster at the same duty cycle (20% exposure, 80% tracking). During tilt, the duty cycle at the exposure region is switched from 20% to 0% by the blocker, see deflector 801 and blocker 802 signals in Figure 8 .

[0089] Scenario C: During acquisition, the beam is kept at the exposure region. This allows using all camera frames during exposure (instead of every other frame), improving the temporal resolution. The deflector signal needs to be switched to constant (not depicted in any figure), so that both the region of interest (ROI) and the tracking region T are imaged simultaneously.

[0090] It is noted that the duty cycles shown in Figs. 6 to Figure 8 and described in scenarios A and BC add up to 100%, but this is not necessarily the case. For example, at the region of interest (ROI), a duty cycle of only 1% can be used (i.e. stroboscopic illumination).

[0091] It is further noted that scenarios A and B can be combined, e.g. changing 5 independent parameters:

[0092] (1) slow (across-frame) period length

[0093] (2) slow duty cycle for the region of interest (ROI);

[0094] (3) slow duty cycle for the tracking region T;

[0095] (4) fast (in-frame) duty cycle for the region of interest (ROI);

[0096] (5) fast (in-frame) duty cycle for the tracking region.

[0097] It can be understood from the above description that the charged particle microscope M is arranged for and the method as described herein comprises tracking the field of view (FOV) during the step of obtaining the tilt series of images by exposing a tracking area substantially outside the region of interest (ROI). The step of obtaining the tilt series of images comprises exposing the region of interest for an exposure step to acquire a tilt series of images, and a tilting step of tilting the sample to a new tilt angle. It can thus be understood that "during the step of obtaining the tilt series of images" can comprise exposing the tracking area T between successive exposures of the region of interest (ROI) at two different tilt angles. It can further be understood that "during the step of obtaining the tilt series of images" can also comprise exposing the tracking area T during the tilting step of tilting the sample to a new tilt angle.

[0098] The method and charged particle microscope M as described herein allow reliably tracking the field of view (FOV) without any overhead.

[0099] The technique can be used for tilt tomography and continuous tilt tomography.

Claims

1. A method comprising: Obtaining tilt series images based on exposing a region of interest (ROI) of the sample to a charged particle beam (CPB) at multiple tilt angles; - Tracking the field of view (FOV) of the region of interest; The feature is that the step of tracking the field of view (FOV) is performed during the step of obtaining the oblique series of images and includes exposing a tracking area substantially outside the region of interest (ROI).

2. The method according to claim 1, comprising the steps of: The charged particle beam is deflected between the region of interest and the tracking region. 3 . The method according to claim 2 , wherein the ratio of the exposure of the region of interest to the exposure of the tracking region is between 1:2 and 1:10, specifically about 1:

4. The method according to claim 1 , wherein the tracking area is completely separated from the region of interest.

5. The method according to claims 1 to 4, comprising the steps of: A field of view shift of the region of interest is determined using the image of the tracking region.

6. The method according to claims 1 to 5, comprising the steps of: Using the field of view of the tracking region provides a feedback loop during the step of obtaining the tilt series of images of the region of interest.

7. The method according to claims 1 to 6, comprising the steps of: Exposure of the region of interest is at least partially prevented using a blocker during the step of exposing the tracking area.

8. The method according to claims 1 to 7, comprising the steps of: The field of view is corrected by moving the sample relative to the charged particle beam.

9. The method according to claims 1 to 8, wherein the method comprises step-wise tomography or continuous tilt tomography.

10. A charged particle microscope, comprising: - a charged particle optical column for directing the charged particle beam onto the sample; - a sample holder for holding a sample; and - a charged particle detector and an imaging system for generating an image signal based on information from the charged particle detector; wherein the charged particle microscope is arranged for - obtaining a tilt series of images based on exposing a region of interest (ROI) of the sample to the charged particle beam (CPB) at multiple tilt angles; - Tracking the field of view (FOV) of the region of interest (ROI); Said feature is that the charged particle microscope M is arranged for tracking the field of view (FOV) by exposing a tracking area substantially outside the region of interest (ROI) during the step of obtaining the tilt series of images.

11. Charged particle microscope M according to claim 10 and arranged for performing the method as defined in claims 1 to 9.

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