Methods for imaging samples using electron microscopy

Abstract

The invention relates to a method for imaging a sample, the sample being mounted on a sample holder in an electron microscope, the electron microscope comprising an electron source for generating a high energy electron beam along an optical axis and optical elements for focusing and deflecting the beam in order to irradiate the sample with the electron beam. The sample holder is capable of positioning and tilting the sample relative to the electron beam. The method comprises the steps of acquiring a series of tilted images by irradiating the sample with the electron beam and simultaneously changing the position of the sample during the acquisition of the images so that each image is acquired with an associated unique tilt angle and an associated unique position.

Description

Technical Field

[0001] The present invention relates to a method for imaging a sample using an electron microscope.

[0002] An example of this approach is available from FEI's "Technical note: Functional description of Tecnai™ Tomography Software", published on December 31, 2003. Background Art

[0003] In an electron microscope, such as a transmission electron microscope (TEM), an electron source generates a high-energy electron beam, the energy of which can be selected, for example, between 80 and 300 keV, although higher and lower energies are known to be used. The beam is manipulated (focused, deflected) by optical elements (electron lenses, deflectors, multipoles) and illuminates a sample placed in a sample holder. The sample holder holds the sample, which typically exhibits three translational degrees of freedom (x, y, z) and one rotational degree of freedom (α) or more. The sample is thin enough to be transparent to electrons: some electrons are absorbed, but many pass through the sample with or without being scattered. Typically, the thickness of the sample is between 50 nm and 1 μm for biological samples (50 and 200 nm for cryogenic samples) and less than 100 nm for semiconductor samples. Another set of optical elements forms a magnified image of the sample on a detector, such as a CMOS camera, a CCD camera, a phosphor screen, or a combination of a phosphor screen and a camera connected by an optical fiber.

[0004] As described in the technical note, the image formed on the detector is a 2D projection of the sample. To obtain a 3D image, a series of images of the sample in different rotation directions are acquired in a so-called tilt series. Typically, the tilt series covers a tilt range from -70 to +70 degrees with regular intervals of 1 or 2 degrees. The images of the tilt series are then aligned to each other and 3D reconstructed using appropriate software and then visualized.

[0005] For each image of the tilt series, the sample must be oriented to another tilt angle. Due to the high resolution of the TEM, the sample should be stable within, for example, 1 nm or less. Therefore, relaxation times of 1 second or longer are usually used to suppress the drift of the stage. As a result, acquiring a tilt series is a time-consuming process.

[0006] In US Pat. No. US 9,147,551 B2 in the name of the present applicant a method of acquiring a tilt series is described wherein the sample holder continuously changes the tilt angle while the tilt series is being acquired and the position of the sample is continuously varied to keep the sample on the optical axis. This so-called "tomo-on-the-fly", made possible by the development of faster acquisition times and smooth tilting mechanisms, reduces the time required to acquire a tilt series.

[0007] It is desirable to further improve methods for acquiring tilt series. Of particular interest is improving the resolution of the images obtained, and / or increasing the speed at which a tilt series can be acquired. Summary of the invention

[0008] To this end, a method for imaging a sample is provided, wherein the method is as defined in claim 1. According to the method, a sample holder is used which is capable of orienting and tilting the sample relative to the electron beam. The method comprises the steps of acquiring a series of tilted images by irradiating the sample with the electron beam, recording the images on a camera at a readout speed, wherein the tilt angle is continuously changed while acquiring the series of tilted images.

[0009] According to a first aspect, the method further comprises a parallel step of changing the position of the sample while acquiring the tilt series. In particular, the position is continuously changed while acquiring a series of tilted images. This means that the position of the sample changes simultaneously with the change of the tilt angle. As a result, each image is acquired with an associated unique tilt angle and an associated unique position.

[0010] Note that in the method described in US 9,147,551 B2 cited above, the position of the sample is continuously changed to keep the sample on the optical axis. Therefore, in practice, there is no actual parallel change in the sample position, because the explicit intention of the described method is to maintain a fixed position during the acquisition of the tilt series. Therefore, this prior art method results in each image being acquired with an associated unique tilt angle and a single fixed position. However, according to the above-mentioned aspects of the present invention, the position is continuously changed during the continuous tilting of the sample to provide a set of unique tilt angles and a set of corresponding unique positions. Therefore, the position of the sample changes, thereby generating additional sample information in the acquired image set. Thus, the purpose of the present invention is achieved.

[0011] Note that changing the position may include the step of increasing the imaged portion of the sample, especially during acquisition of the tilt series. In this way, a larger portion of the sample is imaged while maintaining the desired imaging resolution. In this regard, it is conceivable to change the position by means of a relative movement between the sample and the electron beam. For example, the sample may be moved relative to the optical axis. For example, the position of the sample may be continuously changed by mechanically moving the sample relative to the electron beam. In another embodiment, the electron beam is moved relative to the sample.

[0012] In this regard, it is also noted that it is known to perform a first tilt series, subsequently move the sample to a new position, and then perform a second tilt series at this new position. The new position is chosen in such a way that there is some overlap with the previous position, so that the resulting images for the different tilt series have some overlap and can be stitched together into one or more final images. According to said known method, the movement of the sample is not simultaneous with the acquisition of the tilt series. In fact, the movement of the sample is an intermediate step between the first tilt and the second tilt series.

[0013] According to the method disclosed herein, the change of position is synchronized with the change of the tilt angle. Therefore, no time is wasted in moving and stabilizing the position of the sample holder to a new position. In addition, the camera is continuously acquiring images, making the method disclosed herein also applicable to rapidly changing samples. Another advantage is that all electrons used to irradiate the sample and hit the camera are used for imaging, and therefore no useless sample degradation occurs. In this regard, it is known that in particular biological samples deteriorate due to radiation damage caused by electron irradiation.

[0014] According to another aspect, a method is as defined in claim 5. According to this aspect, the method comprises the step of changing a further tilt angle of the sample in parallel while acquiring the tilt series, such that each image is acquired with an associated unique tilt angle and an associated unique further tilt angle. The further tilt angle may be orthogonal with respect to the tilt angle. Using this method more information about the sample may be generated during a single tilt series, because it reduces the so-called leaky wedge phenomenon. Thereby, the object of the invention is achieved.

[0015] Therefore, it can be concluded from the above that continuously changing the tilt angle and position of the sample, or further changing the tilt angle of the sample, while acquiring a series of tilted images of the sample, respectively, will result in faster acquisition times and / or more information about the sample. Therefore, an improved method for imaging a sample is obtained. Thus, the object of the present invention is achieved.

[0016] Other advantageous embodiments are discussed below.

[0017] In an embodiment, the sample is imaged on a pixelated camera and the speed of the tilt angle expressed in degrees per second, the speed of the parallel change of the position or another tilt angle expressed in meters per second or degrees per second, respectively, and the speed of the camera expressed in frames per second are matched so that the image displacement caused by the tilt change during one frame is, for example, less than one pixel, and preferably less than ½ pixel. By thus matching the tilt speed and the readout speed, the resolution of the camera is optimally used. Known cameras for these purposes include, for example, 4096×4096 pixels. The readout speed of the camera may be limited by the possibilities of the camera or the need for a sufficient number of electrons in one image. Note that it is possible to integrate multiple images, each with a low number of electrons, but this will cause a reduction in the signal-to-noise ratio compared to one image with a longer illumination time and the same number of detection electrons available in the integrated image set. Therefore, the acquisition of one image with a longer frame time is preferred over the integration of multiple images with a lower frame time. This may therefore lead to choosing a readout speed of the camera and a tilting speed of the sample holder, wherein each frame comprises enough electrons, both as many as are required for an image (e.g. for forming a 3D reconstruction (tomogram)) and for acquiring the data required to shift the images relative to each other (using e.g. correlation techniques). In practice, however, the readout speed of the camera is the limiting factor. Therefore, the highest possible frame rate is chosen, and the dose rate and tilting speed are adapted to said readout speed / frame rate.

[0018] In an embodiment, stroboscopic illumination of the sample is used. The sample is illuminated in a stroboscopic manner and the frequency of the illumination pulses can be synchronized with the camera frame readout. Preferably, a single pulse is provided at the beginning of the readout of each frame. The duration of the pulse specifies the amount of motion blur remaining and preferably, a duration is chosen to be as small as technically possible. Due to the stroboscopic illumination, it is possible to acquire high-quality tilt series with an arbitrarily large number of angles at high speed while reducing "smearing" effects caused by motion blur of the image due to tilt increments and / or repositioning during the exposure time of a single acquired image.

[0019] In an embodiment, the speed of the tilt is modulated. The rotation of the sample results in a change in the image. This change is a function of the tilt angle: Δ = d·sinα, where Δ is the image point displacement caused by the rotation, d is the distance of the sample point relative to the tilt axis, and α is the tilt axis. For the same displacement between images, at small tilt angles (sample perpendicular to the optical axis), the tilt speed may therefore be greater than at large tilt angles.

[0020] In an embodiment, a shift and / or rotation correction of the images forming the tilt series is applied relative to each other. The images forming the tilt series should be arranged in a shifted and rotated manner so that one point on the tilt axis serves as a reference for all images. This may include paired corrections. However, if the images include too little information per image for the algorithm used, an algorithm using two or more sets of images may be used, wherein, for example, the shift and / or rotation correction is based on information of two sets each including two images. In addition, it is conceivable that the sample includes markers and that these markers are used for shift and / or rotation correction. By using markers with a diameter of, for example, 10 to 20 nm, such as gold markers, the position and orientation of each image can be found more easily. Preferably, the use of more than one marker enables not only shift correction, but also rotation correction of the images forming the tilt series. Similarly, the tilt angle can be derived from the relative position of a set of markers.

[0021] According to one aspect, there is provided an electron microscope for imaging a sample, wherein the electron microscope comprises:

[0022] - an electron source configured to generate an electron beam along an optical axis of the electron microscope;

[0023] - sample holder;

[0024] - a camera system for acquiring images; and

[0025] - a programmable controller configured to control the sample holder and the camera system, wherein the programmable controller is programmed to control the sample holder and the camera system such that the camera can acquire a series of tilted images of the sample on the sample holder as the sample holder is continuously tilted, and the sample position is changed in parallel while acquiring the tilted series, such that each image is acquired with an associated unique tilt angle and an associated unique position. In an embodiment, the sample holder is capable of tilting the sample and moving the sample relative to the electron beam in parallel. This allows the position of the sample to be changed during sample tilting. In particular, this allows an increase in the imaged portion of the sample during sample tilting.

[0026] According to another aspect, there is provided an electron microscope for imaging a sample, wherein the electron microscope comprises:

[0027] - an electron source configured to generate an electron beam along an optical axis of the electron microscope;

[0028] - sample holder;

[0029] - a camera system for acquiring images; and

[0030] - A programmable controller configured to control the sample holder and the camera system, wherein the programmable controller is programmed to control the sample holder and the camera system so that the camera can acquire a series of tilted images of the sample on the sample holder when the sample holder is tilted continuously around a first tilt axis and continuously around a second tilt axis, so that each image is acquired with an associated unique tilt angle and an associated another unique tilt angle. This allows the sample to be tilted around two different tilt axes: a first axis and a second axis. The two axes can be orthogonal to each other. The two axes can define a plane that is substantially parallel to the plane defined by the sample. The controller can be arranged to change the first tilt angle at a different speed compared to the change in the second tilt angle. It is also possible to assign mutually different amplitudes.

[0031] Note that the electron microscopes according to the two aspects described above can be combined. In other words, the programmable controller can be configured to tilt the sample about a first axis while changing another tilt angle in parallel and changing the position of the sample relative to the electron beam in parallel.

[0032] In all embodiments of the electron microscope, it is conceivable that the electron microscope is programmed with software and that the software enables the electron microscope to perform the methods of imaging a sample described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the method and electron microscope disclosed herein will now be described with reference to the accompanying drawings, in which:

[0034] Figure 1 - schematically depicts a transmission electron microscope;

[0035] Figure 2 - shows a typical workflow for producing an image of a sample, which may be a tomogram;

[0036] Figures 3a to 3b - illustrates an embodiment of a schematic scanning pattern according to the first aspect of the method disclosed herein, wherein the tilt angle and the position movement are applied successively;

[0037] Figures 4a to 4c - show further embodiments of scanning patterns according to the method;

[0038] Figure 5 - showing another scanning mode according to the method;

[0039] Figure 6 - A graph showing another aspect of the method as disclosed herein, wherein a first tilt angle period and a further tilt angle period are applied. DETAILED DESCRIPTION

[0040] 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 specifically, it shows an embodiment of a transmission type microscope M, which in this case is a TEM / STEM. Note that in the context of the present invention, it could equally effectively be a SEM or an ion-based microscope, for example.

[0041] exist Figure 1 In the figure, within a vacuum housing 2, an electron source 4 generates an electron beam B, which propagates along an electron optical axis B' and passes through an electron optical illuminator 6, thereby being used to guide / focus the electrons onto a selected portion of a specimen S (e.g., which can be (locally) thinned / planarized). A deflector 8 is also depicted, which can be used to achieve a scanning motion of the electron beam B.

[0042] The specimen S is held on a specimen holder H, which can be positioned with multiple degrees of freedom by a positioning device / platform A, which moves a bracket A' to which the holder H is attached. The specimen holder H may include fingers that can move in the XY plane (see Figure 1 The specimen holder H is a Cartesian coordinate system in the embodiment of the present invention, and in general, movement parallel to Z and tilting about X and / or Y will also be possible. Such movement allows different parts of the specimen S to be illuminated / imaged / inspected by the electron beam B traveling along the axis B' (in the Z direction). As an alternative to beam scanning, the movement also allows a scanning movement to be performed. If necessary, an optional cooling device (not depicted) can be brought into close thermal contact with the specimen holder H in order to maintain it (and the specimen S thereon) at a low temperature.

[0043] The electron beam B will interact with the specimen S so that various types of "stimulated" radiation are emitted from the specimen S, including (for example) secondary electrons, backscattered electrons, X-rays, and optical radiation (cathodoluminescence). Electrons that pass through the specimen S and continue to propagate along the axis B' can be studied. This transmitted electron flux enters an imaging system 24, which will typically include various electrostatic / magnetic lenses, deflectors and / or correctors. In normal (non-scanning) TEM mode, this imaging system 24 can focus the transmitted electron flux onto a fluorescent screen 26, which can be retracted / retracted (as schematically indicated by arrow 26') to move it away from the axis B' as required. An image (or diffraction pattern) of (a portion of) the specimen S will be formed by the imaging system 24 on the screen 26, and this can be viewed through an observation port 28 located in an appropriate portion of the wall of the housing 2. For example, the retraction mechanism of the screen 26 is mechanical and / or electrical in nature and is not depicted here.

[0044] As an alternative to viewing the image on screen 26, one may instead exploit the fact that the depth of focus of the electron flux leaving the imaging system 24 is typically very large (e.g., about 1 meter). Thus, various other types of analysis equipment may be used downstream of screen 26, such as:

[0045] - TEM camera 30. At camera 30, the electron flux may form a static image (or diffraction pattern) which may be processed by controller / processor 20 and displayed on display device 14, such as, for example, a flat panel display. When not needed, camera 30 may be retracted / retracted (as schematically indicated by arrow 30') so that it is away from axis B';

[0046] - STEM detector 32. The output from detector 32 can be recorded as a function of the (X,Y) scanning position of electron beam B on specimen S, and an image can be constructed, which is a "map" of the output from detector 32 as a function of X,Y. Detector 32 may include a single pixel of, for example, 20 mm in diameter, as opposed to the matrix of pixels characteristically present in camera 30. Furthermore, detector 32 will typically have a higher resolution than camera 30 (e.g., 10 pixels per second). 2 images per second) 6 Likewise, when not needed, the detector 32 may be retracted / retracted (as schematically indicated by arrow 32') to move it away from the axis B'.

[0047] As an alternative to imaging using camera 30 / detector 32 , it is also possible to call upon a spectroscopic device 34 , 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 critical and many possible variations are envisioned. For example, spectroscopic device 34 may also be integrated into imaging system 24 .

[0049] In the illustrated embodiment, microscope M also includes a retractable computed tomography (CT) module, generally indicated by reference numeral 40. In computed tomography (also known as tomographic imaging), a specimen is observed along different lines of sight using a source (diametrically opposed) detector to obtain a penetrating view of the specimen from various angles. CT can be accomplished by acquiring a tilt series (detecting (S)TEM images and / or X-ray maps at each tilt). X-rays can be detected with detectors 22 and / or 40, with the difference that detector 40 collects a larger solid angle and is therefore faster.

[0050] Note that a controller (computer processor) 20 is connected to the various illustrated components 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 the display device (14). Needless to say, the (schematically depicted) controller 20 may be (partially) inside or outside the housing 2 and may have an integral or composite structure as desired.

[0051] Notice, Figure 1 The electron microscope depicted in includes many components, many of which are described only for the purpose of clarity. Figure 1 The electron microscope 1 shown in FIG. 1 is equipped for imaging a sample, for example using electron tomography, and comprises:

[0052] an electron source 4 configured to generate an electron beam B along an optical axis B′ of the electron microscope 1 ;

[0053] - sample holder H;

[0054] - a camera system 30 for acquiring images; and

[0055] - a programmable controller 20 configured to control the sample holder H and the camera system, wherein the programmable controller 20 is programmed to control the sample holder H and the camera system 30 so that the camera can acquire a series of tilted images of the sample S on the sample holder H while the sample holder H is continuously tilted, and the sample position is changed in parallel while acquiring the tilt series so that each image is acquired with an associated unique tilt angle and an associated unique position. As an alternative to changing the position, the other tilt angle can be changed in parallel while acquiring the tilt series so that each image is acquired with an associated unique tilt angle and an associated other unique tilt angle. In another embodiment, the position and the other tilt angle are changed in parallel to obtain the tilt series so that each image is acquired with an associated unique tilt angle, an associated other unique tilt angle and an associated unique position.

[0056] In an embodiment, the electron microscope is a transmission electron microscope and a TEM camera 30 is used as the camera system. However, in other embodiments, it is conceivable that a STEM detector 32 is used as the camera system instead.

[0057] therefore, Figure 1A TEM 1 is schematically shown. A particle source 4 generates an electron beam along an optical axis B'. The electrons have a selectable energy typically between 80 and 300 keV, although higher energies, such as 400 keV to 1 MeV, or lower energies, such as 50 keV, may be used. The electron beam is manipulated by a condenser system 6 to form a parallel beam that impinges on a sample S, which is positioned on a sample holder H. The sample holder H may position the sample S relative to the optical axis B', and may displace the sample in a plane perpendicular to the optical axis B' (i.e., an XY plane), and may tilt the sample relative to at least one tilt axis (i.e., X and / or Y) perpendicular to the optical axis B'. An imaging system 24 is arranged to form an enlarged image of the sample on a pixelated detector 32, thereby revealing sample details of, for example, 0.1 nm. The detector 32 may take the form of, for example, a CCD or CMOS camera.

[0058] The sample holder H and the camera system 32 are controlled by a controller 20, and the controller may be equipped to convert the tilt series into 3D tomograms and to visualize the tomograms on the screen 14. Note that for the visualization, dedicated software is used, such as described in the aforementioned technical note "Functional Description of the Tecnai™ Tomography Software".

[0059] The controller 126 is programmed to store the images acquired via the camera system 124 in a memory, while controlling the sample holder 110 to tilt around the tilt axis 114 at a controlled tilt speed, and simultaneously controlling the electron microscope to change the orientation of the sample S. The sample stage is capable of tilting the sample at a large angle (typically −80 to +80 degrees) in order to obtain sufficient data to reconstruct a 3D image of the sample. In one embodiment, the sample stage H is capable of changing the orientation of the sample by tilting the sample around another tilt axis, which is particularly perpendicular to the aforementioned tilt axis. Therefore, in an embodiment, the sample stage H can be tilted around the X-axis and the Y-axis.

[0060] In an embodiment, the controller is capable of controlling the position of the sample S relative to the optical axis B' in order to reposition the sample S relative to the optical axis. This may be done by controlling one or more deflectors 8 or by mechanically moving the sample S relative to the fixed optical axis B' by correspondingly controlling the sample holder H.

[0061] According to the method disclosed herein, the orientation of the sample is changed in parallel while acquiring a series of tilted images so that each image is acquired with an associated unique tilt angle and an associated unique orientation. Figure 2 and Figure 3 explain this in more detail.

[0062] Figure 2 A typical workflow for producing an image of a sample using an embodiment of a method as disclosed herein is shown.

[0063] In step 301, a sample is placed in a TEM.

[0064] In step 302, a volume of interest is determined. This may involve the location of a marker. The TEM can be optimally focused on the volume of interest. The defocus is set to obtain an optimum between contrast and resolution.

[0065] In step 303, the sample is placed in a starting position in which a portion of the volume of interest is imaged. The sample is rotated to its first (extreme) tilt angle, e.g., -70 degrees. The sample is then ready for the tilt series and acquisition can begin. Of course, there may be other starting angles, e.g., zero tilt.

[0066] In step 304, the tilt series is started and the tilt is gradually but continuously changed to its other extreme tilt angle, for example +70 degrees. In step 305, while performing step 304, the position of the sample is continuously changed. In this way, different sub-portions of the volume of interest can be imaged. The imaged portion can be kept (defocused) by (additional) x / y / z translation. Steps 304 and 305 can be repeated (as indicated) until the final position is reached in step 306. While changing the position of the sample, the tilt may be gradually changed back and forth, i.e. from -70 degrees to +70 degrees and then back to -70 degrees again. Therefore, multiple tilt cycles can be performed during parallel position changes from the starting position to the final position.

[0067] Once all images have been acquired, the images forming the tilt series are aligned in step 307. Note that this may already have begun when the images are acquired.

[0068] The tilt series is used to compute a tomogram in step 308. Note that this step is started while information is still being acquired, thereby shortening the overall time required.

[0069] In step 309 , the tomogram is visualized based on the user input (desired slice plane, angle, thickness, contrast, etc.).

[0070] It will be apparent to those skilled in the art that this is just one of many implementations of the workflow. For example, the workflow may be modified, or other steps may be added.

[0071] As an example, it can be envisaged that during a continuously varying tilt angle, a sequence of focus settings is applied in parallel. In particular, this means that during a continuously varying tilt angle, the focus may also be continuously changed. Preferably, the speed of changing the tilt angle is relatively low compared to the speed of changing the focus settings, so that within a relatively small tilt range, a cycle of focus settings can be applied. Then, within a next relatively small tilt range, another cycle of focus settings is applied again. By capturing a corresponding sequence of component images within each (relatively small) sample tilt angle range, it can be ensured that subsequent images within a given tilt angle range have multiple parts of the sample with a given desired focus state (e.g. in focus or a given degree of defocus). It is known to the person skilled in the art that, in principle, for a tilted sample in a given focus state, only one y-coordinate on the sample will have said state (see Figure 1 ). Thus, in prior art transmission tomography, a mathematically reconstructed image of a given (surface) "layer" or "level" of a sample will typically exhibit poor resolution and an unstable power spectrum (essentially due to processing the data by using mixed degrees of "blurring". However, if the focus setting is changed while continuously tilting the sample, a 3D imaging cube can be captured which will allow reconstruction of multiple y-coordinates on the sample to select the focus state. Note that it is known from US 8,912,491 B2 in the name of the applicant to acquire a sequence of focus settings at fixed tilt angles and to repeat the sequence for each fixed tilt angle within a range of discrete tilt angle intervals.

[0072] In particular, it is conceivable to interchange the position and the tilt angle. This means that, as before, a starting tilt angle and a starting position are selected in step 303, and this tilt angle is continuously changed in step 305 until the end tilt angle is reached in step 306. At the same time, the sample position is changed from the starting position to the end position and then back to the starting position again to perform one or more further cycles from the starting position to the end position. Thus, multiple cycles of position movement can be performed during the parallel change of the tilt angle, which can be a single cycle from, for example, -70 degrees to +70 degrees, or also multiple cycles.

[0073] Figure 3 to Figure 6 Possible embodiments of scanning modes according to the methods disclosed herein are given.

[0074] Figure 3a A schematic scanning pattern with associated tilt angle and sample position relative to the electron beam is shown. Figure 3b Shown with Figure 3a A portion of the scan pattern corresponding to the position (or time) of the sample holder and the tilt angle is shown in the figure.

[0075] exist Figure 3a In FIG. 1 , V denotes the volume of interest of the sample to be imaged. The size of the volume of interest V (solid square) is larger than the available field of view C (dashed line) of the camera. Here, the volume of interest V is approximately equal to nine times the field of view C. According to the prior art method, each subsection C is visited once and a series of oblique images are obtained for the respective subsection C. Some overlap is required between the subsections in order to be able to stitch the subsequent images together.

[0076] According to one aspect of the method disclosed herein, Figure 3a As shown in FIG. , the tilt angle of the sample holder changes from the initial tilt position t s To the middle tilt position t m and the end tilt position t e Continuous change. Once the end tilt position t is reached e , the sample is moved back to start tilting again at angle t s In the embodiment shown ( Figure 3b ), the backward tilt occurs at a very fast speed, but in practice the speed is limited by the possibilities of the platform.

[0077] At the same time, while continuously changing the tilt angle, the position of the sample relative to the electron beam also changes. s Start and zigzag toward the end position P e Move, such as Figure 3a The tilting speed and the relative movement of the sample are chosen here so that all tilt positions are visible in a single subsection. In practice, this means that a specific feature of the sample will be visible at, for example, a tilt angle t s enters the image in the left part and gradually moves through the image at a moving speed until it is tilted, for example, at an angle t e The image is left in the right part. For multiple cycles of tilting, the tilting speed can of course be increased to make specific features visible in the image.

[0078] like Figure 3a and Figure 3b The effect of the positioning and tilting motion shown is that each image is acquired with an associated unique tilt angle and an associated unique position, and the volume of interest V of the sample can be rapidly imaged while preserving detail associated with the particular field of view C.

[0079] Of course, it will be clear to those skilled in the art that the positioning pattern and the tilting pattern may be modified as required. Figure 4a and Figure 4b Two embodiments are shown, in which different movement patterns are used (from the starting position P s To the end position P e), and wherein the rocking back-and-forth motion is used to tilt the angle (see also Figure 4c , which shows a graph of time or position versus tilt angle.

[0080] Figure 5 An embodiment is shown in which the positioning movement is relatively fast compared to the tilting speed. s To the end position P e The tilt angle is measured as Δα from t s Change to t2. Then, reposition the sample to the starting position P again. s and forms another trajectory in which the sample is tilted by an additional amount Δα. The process is repeated until a point is reached from t s to e The desired tilt range can be, for example, -70 degrees to +70 degrees. Of course, other ranges are also conceivable.

[0081] Figure 6 An embodiment is shown in which a first (fixed) tilt angle varies continuously (dashed line) and at the same time a second (fixed) tilt angle also varies (solid line). The second tilt axis may be substantially orthogonal to the first tilt axis. The same rate of variation may be selected for the first and second tilt angles, but in the embodiment shown the rate of variation of the first tilt angle (dashed line) is very low compared to the rate of variation of the second tilt angle (solid line). For the first tilt angle, the tilt range may be -70 degrees to +70 degrees, and for the second tilt angle, -10 degrees to +10 degrees. Other values ​​are also conceivable. For a relatively small tilt range of the first angle, the entire range of motion of the second tilt angle may be achieved. By way of example, a variation of the second tilt angle from -10 degrees to +10 degrees may be achieved while the first angle changes by approximately 5 degrees. Thus, within a given range of -70 degrees to +70 degrees, the range of the second tilt angle is achieved (140 divided by 5 equals) 14 times. In Figure 6 In the embodiment shown in , for a single cycle of the first tilt angle from -70 degrees to +70 degrees, the second tilt angle is completed a total of 25 times. It will be understood that other values ​​are also conceivable. Note that, in addition, a change in position (as with respect to the previous FIGS. 3 to 4) may also be applied. Figure 5 The stages for achieving such a tilting movement are known per se to a person skilled in the art.

[0082] The protection desired is determined by the following claims.

[0083] Non-patent literature

[0084] [-1-]Technical Note: “Functional Description of Tecnai™ Tomography Software”, FEI Company, version 31 December 2003.

Claims

1. A method of imaging a sample, the sample being mounted on a sample holder in an electron microscope, the electron microscope comprising an electron source for generating a high energy electron beam along an optical axis and optical elements for focusing and deflecting the beam so as to illuminate the sample with the electron beam, the sample holder being capable of positioning and tilting the sample relative to the electron beam, the method comprising: - acquiring a series of tilted images by irradiating the sample with the electron beam, recording the images on a camera at a readout speed, wherein the tilt angle is continuously varied while acquiring the series of tilted images; and - changing the position of the sample while acquiring the series of tilted images such that each image is acquired with an associated unique tilt angle and an associated unique position, wherein a plurality of tilt cycles are performed during a parallel position change from a starting position to a final position.

2. The method of claim 1, wherein changing the position comprises increasing the imaged portion of the sample.

3. A method according to claim 1 or 2, wherein the position is changed by relative movement between the sample and the electron beam. The method of claim 3 , wherein the position of the sample is continuously changed by mechanically moving the sample relative to the electron beam.

5. A method of imaging a sample, the sample being mounted on a sample holder in an electron microscope, the electron microscope comprising an electron source for generating a high energy electron beam along an optical axis and optical elements for focusing and deflecting the beam so as to irradiate the sample with the electron beam, the sample holder being capable of positioning and tilting the sample relative to the electron beam, the method comprising: - acquiring a series of tilted images by irradiating the sample with the electron beam, recording the images on a camera at a readout speed, wherein the tilt angle is continuously varied while acquiring the series of tilted images; and - varying a further tilt angle of the sample while acquiring the series of tilted images, such that each image is acquired with an associated unique tilt angle and an associated further unique tilt angle. The method of claim 5 , wherein the further tilt angle is orthogonal to the tilt angle.

7. A method according to claim 5 or 6, wherein the sample is imaged on a pixelated camera, and the speed of the tilt angle expressed in degrees per second, the speed of the respective parallel changes of the position or another tilt angle expressed in meters per second or degrees per second, respectively, and the readout speed of the camera expressed in frames per second are matched so that the displacement of the image caused by the tilt change during one frame is less than one pixel.

8. A method according to claim 5 or 6, wherein stroboscopic illumination of the sample is used.

9. The method according to claim 5 or 6, wherein A displacement and / or rotation correction is applied which forms the series of tilted images relative to each other.

10. The method of claim 9, wherein the displacement and / or rotation correction is based on one or more of the following: - Algorithms that use data from more than two images to simultaneously correct the relative positions of multiple images; - Pairwise correction based on an algorithm using data from two images; - The location of one or more markers in the image.

11. An electron microscope equipped for imaging a sample, the electron microscope comprising: - an electron source configured to generate an electron beam along an optical axis of the electron microscope; - sample holder; - Camera system for acquiring images; and - a programmable controller configured to control the sample holder and the camera system, wherein the programmable controller is programmed to control the sample holder and the camera system so that the camera is able to acquire a series of tilted images of the sample on the sample holder as the sample holder is continuously tilted, and the sample position is changed while acquiring the series of tilted images so that each image is acquired with an associated unique tilt angle and an associated unique position, wherein multiple tilt cycles are performed during a parallel position change from a starting position to a final position.

12. An electron microscope equipped for imaging a sample, the electron microscope comprising: - an electron source configured to generate an electron beam along an optical axis of the electron microscope; - sample holder; - Camera system for acquiring images; and - a programmable controller configured to control the sample holder and the camera system, wherein the programmable controller is programmed to control the sample holder and the camera system so that the camera is able to acquire a series of tilted images of the sample on the sample holder when the sample holder is tilted continuously about a first tilt axis and continuously about a second tilt axis, so that each image is acquired with an associated unique tilt angle and an associated another unique tilt angle.

13. An electron microscope according to claim 11 or 12, wherein the electron microscope is programmed with software, and wherein the software enables the electron microscope to perform a method of imaging a sample as defined in one or more of claims 1 to 9.

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