Method for imaging a sample using a transmission charged particle microscope

Abstract

The present disclosure relates to a method of imaging a sample using a transmission charged particle microscope, the method comprising providing a sample, and providing a charged particle beam and directing the charged particle beam onto the sample for generating a flux of charged particles transmitted through the sample. The method comprises the step of generating and recording a first energy filtered flux of charged particles transmitted through the sample, wherein the first energy filtered flux of charged particles consists essentially of unscattered and elastically scattered charged particles. The method as disclosed herein comprises the further step of generating and recording a second energy filtered flux of charged particles transmitted through the sample, wherein the second energy filtered flux of charged particles consists essentially of inelastically scattered charged particles. Then, the first and second recorded energy filtered fluxes are used for imaging the sample with increased contrast.

Description

[0001] manual The present invention relates to a method for imaging a sample using a transmission charged particle microscope, the method comprising providing a sample and providing a beam of charged particles, and guiding the beam of charged particles onto the sample to generate a certain flux of charged particles that are transmitted through the sample.

[0002] Charged particle microscopy, particularly in the form of electron microscopy, is a well-known and increasingly important imaging technique for microscopic objects. Historically, the basic type of electron microscope has evolved into several well-known instrument types, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning transmission electron microscopy (STEM), and has also evolved into various subtypes, such as so-called "dual-beam" instruments (e.g., FIB-SEM), which additionally employ a focused ion beam (FIB), thereby allowing for activities such as ion beam milling or ion beam induced deposition (IBID). Technicians will be familiar with different types of charged particle microscopy.

[0003] In TEM, the electron beam used to irradiate the sample is chosen to have sufficiently high energy to penetrate the sample (for this purpose, the sample will typically be thinner than in the case of a SEM sample); the flux of transmitted electrons emitted from the sample can then be used to create an image. When this type of TEM operates in scanning mode (and thus becomes STEM), the image in question is accumulated during the relative scanning motion of the electron beam and the sample.

[0004] There are several use cases in transmission electron microscopy (TEM) in which an electron beam moves or scans over a sample.

[0005] One example is single-particle analysis (SPA). In this workflow, the sample contains a grid with numerous circular apertures, each aperture containing a piece of ice foil with a copy of the biological particle to be imaged. Each ice foil is approximately 2 μm in diameter, and the foils are spaced approximately 5 μm apart. The stage is moved to the center of the apertures, and 2 to 6 images are acquired using 2 to 6 different beam-image offsets of approximately 0.5 μm, each image covering approximately 0.5 x 0.5 μm. 2 The area. In this case, beam-image offset refers to the combined deflection of the illumination beam above the sample and the image beam below the sample, such that after two deflections, the beams are coaxial in the imaging system downstream of the sample, and that a portion of the sample that is not coaxial is imaged. The stage then moves to the next aperture (typically 5 μm away) and the procedure is repeated. This process can be repeated hundreds or even thousands of times to generate multiple images.

[0006] In single-particle analysis (SPA), the 3D structure of a biological particle, such as a protein or virus, is reconstructed from multiple images, where each individual image may contain dozens of copies of the same biological particle. One step in this process involves identifying and locating particles in multiple images. These particles have (very) low contrast because both the particle and the ice they are embedded in are composed of light elements (N, C, O, H). This makes it difficult to identify and characterize the particles in the images.

[0007] Traditional methods to enhance contrast include defocusing (CTF theory teaches that this increases the transmission of low spatial frequencies) or applying phase plates. The first method has the drawback of reducing information at high resolution. The latter has the drawback that, in practice, all available phase plates will block some portion of the beam intensity.

[0008] Therefore, the object of the present invention is to provide a method for increasing the contrast of images obtained in a transmission charged particle microscope.

[0009] For this purpose, the present invention provides a method for imaging a sample using a transmission charged particle microscope as defined in claim 1.

[0010] The method as defined herein is characterized by comprising the steps of: generating and recording a first energy filtering flux transmitted through charged particles in the sample, wherein the first energy filtering flux of the charged particles consists substantially of unscattered and elastically scattered charged particles. The first energy filtering flux of the charged particles enhances the contrast between different regions in the sample, such as the contrast between particles and the ice embedded within them.

[0011] The method as defined herein is further characterized by comprising the steps of: generating and recording a second energy filtering flux of charged particles transmitted through the sample, wherein the second energy filtering flux of the charged particles consists primarily of inelastically scattered charged particles. Since the differences between different regions in the ratio of inelastically scattered charged particles to unscattered or elastically scattered charged particles are small but significant, the second energy filtering flux of the charged particles contains information about different regions of the sample, such as particles and the ice embedded within them, and this can be used to further improve the contrast of the image obtained by this method.

[0012] This method combines information from the energy filtering flux of the first and second records to image the sample with increased contrast. Thus, the objective of the invention is achieved.

[0013] Further embodiments and advantages will be described below.

[0014] The method defined in this paper is based on the insight that there are small but significant differences in the ratio of inelastically scattered charged particles to those that are not scattered or are elastically scattered between different regions of a sample. This is, for example, the case between ice and particles in a SPA sample. Here, inelastic scattering means that charged particles in a beam, when scattered on the sample, transfer a portion of their kinetic energy to the sample not only due to momentum transfer (which is negligible in the case of charged particles being electrons, due to the large mass difference between electrons and atoms in the sample), but also due to internal excitations of electrons or atoms in the sample. Such internal excitations can include (in decreasing order of energy transfer) so-called core losses (which occur when bound electrons in the core-shell of atoms in the sample are excited into the shell or vacuum), or plasmon excitations (which are collective excitations of valence electrons), or bandgap transitions (when electrons in the valence band are excited into the conduction band), or phonon excitations (which are collective vibrations of atoms in the sample). Typical energy transfer associated with these processes are: 100 eV to 2000 eV for core excitation, 10 eV to 40 eV for plasmon excitation, 2 eV to 6 eV for bandgap transition, and 0.01 eV to 0.2 eV for phonon excitation.

[0015] In contrast, elastic scattering refers to the phenomenon where charged particles in the beam, when scattered across the sample, do not induce any internal excitation of electrons or atoms within the sample. It can be noted that for thin samples (10 nm…30 nm) typically used in transmission electron microscopy, most electrons in the illumination beam are not scattered or elastically scattered with the sample. Only a small fraction (typically 5%…20%) of the electrons undergo inelastic scattering, and most of these inelastic scattering events are plasmon excitations.

[0016] Typically, the combined flux of unscattered charged particles and elastically scattered charged particles is called the elastic signal, elastic flux, or elastic profile.

[0017] Furthermore, this method employs spectroscopic equipment to analyze the energy transferred or lost by charged particles when interacting with a sample, and specifically, to create images of the sample using only charged particles that have experienced a specific energy loss. In the context of electron microscopy, such equipment is often referred to as an electron energy loss spectroscopy (EELS) module.

[0018] The first energy filtering flux can be a so-called zero-loss peak (ZLP) filtered image, in which all electrons that induce some energy loss in the sample are filtered out of the image. The prior art understanding is that these electrons that have already induced energy loss (inelastic scattering electrons) are out of focus in the image due to energy differences, and this out-of-focus signal gives the image a blurred background. This blurred background reduces the signal-to-noise ratio and thus reduces the visibility of particles in the ice. According to this prior art understanding, ZLP filtering would improve the contrast of thick samples (e.g., >100 nm) in which significant inelastic scattering occurs (say, >50%), but not for thin samples (e.g., <30 nm) in which only a small amount of inelastic scattering occurs (say, <20%). However, the inventors have found that even for very thin samples (<30 nm), ZLP filtering can improve contrast. As will be shown later, the improvement in SPA contrast obtained by ZLP filtering does not come from removing the blurred background, but from the significant difference in inelastic scattering between the particles and the surrounding ice in which they are embedded.

[0019] The second energy filtering flux can be an image blocked by a so-called zero-loss peak (ZLP). The inventors discovered that ZLP-blocked images do indeed carry some compelling information. For example, in SPA, the inelastic signal is higher at the particle's location. This increase in signal can be used to enhance image contrast. Logically, this increase in inelastic signal strength is accompanied by a corresponding decrease in elastic signal strength. This decrease in strength (along with high-frequency information) makes the particle identifiable in the ZLP-filtered image.

[0020] In one embodiment, a first dose is used during the step of generating and recording the first energy filtering flux of the charged particles.

[0021] The first dose can be equal to the maximum dose the sample can withstand before losing structural integrity, for example, 40 electrons / Å. 2 .

[0022] In an embodiment, a second dose is used during the step of generating and recording the second energy filtering flux of the charged particles. The second dose may be different from the first dose, but in an embodiment it may also be equal to it.

[0023] In one embodiment, the step of generating and recording a first energy filtering flux of charged particles is performed first, and the step of generating and recording a second energy filtering flux of charged particles is performed later.

[0024] Here, it is conceivable that the second dose is at least equal to the first dose, for example, at least 40 e / Å. 2 .

[0025] The recorded second energy filtering flux of charged particles can be used to locate the region of interest within the sample. If the sample contains multiple sample particles, the region of interest can be the sample particles themselves. For example, sample particles can be embedded in ice.

[0026] In an embodiment, the method may include the step of subtracting a recorded second energy filtering flux of charged particles from a recorded first energy filtering flux of charged particles. This may include the steps of: forming an image of the first and / or the second energy filtering flux of charged particles, and subtracting the image. Other subtraction methods are also conceivable.

[0027] During the subtraction, a scaling factor can be used on the second energy filtering flux of the recorded charged particles.

[0028] As previously described, the sample may include multiple sample particles. In an embodiment, the method includes the step of forming a three-dimensional (3D) reconstruction of the sample particles, wherein the first and second energy fluxes of the charged particles are used.

[0029] According to one aspect, a spectroscopic device as defined in claim 13 is provided. The spectroscopic device as defined herein comprises: a dispersive apparatus for receiving a flux of charged particles and dispersing the flux of charged particles according to their energy loss; and a detection system for detecting at least a portion of the dispersed flux of charged particles.

[0030] As defined herein, spectroscopic devices are arranged for: - Generate and record the first energy filtering flux of charged particles, which consists primarily of unscattered and elastically scattered charged particles; and - Generate and record the second energy filtering flux of charged particles, which consists primarily of charged particles that are inelastically scattered.

[0031] The first energy filter flux and the second energy filter flux can be used to create images of the sample. In an embodiment, the spectroscopic device can be arranged to create such images.

[0032] Thus, as described above, the contrast between particles in the sample and their surrounding environment in the image created from the detected flux can be increased.

[0033] The spectroscopic device may include one or more slit elements for generating a second energy filtering flux for the first and / or charged particles.

[0034] According to one aspect, a transmission charged particle microscope is provided. The transmission charged particle microscope includes: - Charged particle beam source, used to emit charged particle beams; - Sample holder, used to hold samples in place; - An irradiator for guiding a beam of charged particles emitted from a charged particle beam source onto a sample; and - Control unit, used to control the operation of the transmission charged particle microscope.

[0035] As defined in this paper, the transmission charged particle microscope is configured for: - Generate and record the first energy filtering flux of charged particles, which consists primarily of elastically scattered charged particles; and - Generate and record the second energy filtering flux of charged particles, which consists primarily of charged particles that are inelastically scattered.

[0036] Transmission charged particle microscopy may include spectroscopic devices as defined herein.

[0037] The methods, detectors, and microscopes as defined herein will now be illustrated in more detail based on exemplary embodiments and the accompanying schematic diagrams, wherein: Figure 1 A longitudinal cross-sectional view of a charged particle microscope is shown. Figure 2 A detailed view of the spectral device as defined herein is shown in an unfiltered setting; Figure 3 This is shown in the first energy filtering setting as defined herein. Figure 2 Spectroscopic equipment; Figure 4 This shows the second energy filtering setup as defined herein. Figure 2 Spectroscopic equipment; Figures 5a-5c This shows how to use the corresponding different settings. Figure 2-4 Images obtained from spectral equipment; Figure 5d Shown in Figures 5b-5c The combination of images obtained, where Figure 5c Add to Figure 5b middle; Figure 6 A schematic diagram showing the total elastic and inelastic signals within a sample, including the ice layer and the particles embedded therein; Figure 7 Shown in Figures 5b-5c The combination of images obtained from, where from Figure 5b Subtracted from Figure 5c .

[0038] In the figures, corresponding parts are indicated using corresponding reference symbols when relevant. It should be noted that, generally, the figures are not drawn to scale.

[0039] Figure 1 This is a highly schematic depiction of an embodiment of a transmission charged particle microscope M, in which case the transmission charged particle microscope M is a TEM / STEM (however, in the context of this disclosure, it may properly be, for example, an ion-based microscope or a proton microscope). Figure 1 Within a vacuum enclosure E, an electron source 4 (e.g., a Schottky emitter) generates an electron beam (B) that traverses an electron optical illuminator 6, which is then directed / focused onto a selected portion of the sample S (which may, for example, be (locally) thinned / planarized). This illuminator 6 has an electron optical axis B' and typically includes various electrostatic / magnetic lenses, (scanning) deflectors D, correctors (e.g., astigmatism correctors), etc.; it may also typically include a condenser system (the entire object 6 is sometimes referred to as the "condenser system").

[0040] The sample S is held in a sample holder H. As illustrated herein, a portion of this holder H (inside the housing E) is mounted in a bracket A', which can be positioned / moved in multiple degrees of freedom by a positioning device (stage) A; for example, the bracket A' can be (in particular) movable in the X, Y, and Z directions (see the depicted Cartesian coordinate system) and can rotate about a longitudinal axis parallel to X. Such movement allows different portions of the sample S to be irradiated / imaged / detected by an electron beam traveling along axis B' (and / or, for example, allows scanning motion to be performed as an alternative to beam scanning [using (one or more) deflectors D], and / or allows selected portions of the sample S to be processed by a (not depicted) focused ion beam).

[0041] The (focused) electron beam B, traveling along the axis, interacts with the sample S, causing various types of "stimulated" radiation to emanate from the sample S, including (e.g.) secondary electrons, backscattered electrons, X-rays, and optical radiation (catholuminescence). If desired, one or more of these radiation types can be detected by means of a detector 22, which can be, for example, a combination of scintillator / photomultiplier tubes or an EDX (energy-dispersive X-ray spectroscopy) module; in this case, the image can be constructed using essentially the same principles as in SEM. Alternatively or additionally, however, electrons that traverse (through) the sample S, are emitted from it, and continue to propagate along axis B' (essentially, although generally with some deflection / scattering) can be studied. This transmitted electron flux enters the imaging system (combined mirror / projection lens) 24, which will typically include various electrostatic / magnetic lenses, deflectors, correctors (e.g., astigmatism correction devices), etc.

[0042] In normal (non-scanning) TEM mode, this imaging system 24 focuses the transmitted electron flux onto a fluorescent screen 26, which, if desired, can be retracted / withdrawn (as schematically indicated by arrow 26') away from the path of axis B'. An image (or diffraction pattern) of a portion of sample S is formed by the imaging system 24 on screen 26, and this can be observed through a viewing port 28 located in a suitable portion of the wall of housing E. The retraction mechanism of screen 26 may be, for example, mechanical and / or electrical in nature, and is not depicted herein.

[0043] As an alternative to viewing the image on screen 26, the fact that the focusing depth of the electron flux emanating from imaging system 24 is typically quite large (e.g., approximately 1 meter) can be utilized. Therefore, various types of sensing / analysis devices can be used downstream of screen 26, such as: - TEM camera 30. At camera 30, the electron flux can form a still image (or diffraction pattern), which can be processed by controller C and displayed on a display device (not depicted), such as a flat panel display. When not needed, camera 30 can be retracted / withdrawn (as schematically shown by arrow 30') to remove it from axis B'.

[0044] - STEM recorder 32. The output from recorder 32 can be recorded as a function of the (X,Y) scan position of beam B on sample S, and can construct an image, which is a "map" of the output from recorder 32 as a function of X and Y. Recorder 32 may include single pixels with a diameter of, for example, 20 mm, rather than a pixel matrix characteristically present in camera 30. Furthermore, recorder 32 will typically have a larger sensor size than camera 30 (e.g., 10 Hz). 2 A much higher acquisition rate (e.g., 10 images per second) 6 (Points). Again, when not needed, the recorder 32 can be retracted / withdrawn (as schematically indicated by arrow 32') so as to move away from the path of axis B' (but such retraction would not be necessary in the case of, for example, annular dark field recorders 32; in such recorders, the central aperture would allow the beam to pass through when the recorder is not in use).

[0045] - As an alternative to using camera 30 or recorder 32 for imaging, a spectroscopic device 34 may also be invoked, which may be, for example, an EELS module.

[0046] It should be noted that the order / orientation of objects 30, 32, and 34 is not strict, and many possible variations are conceivable. For example, the spectroscopic device 34 may also be integrated into the imaging system 24.

[0047] Note that the controller (which may be a combination of controller and processor) C is connected to the various described components via control lines (buses) C'. The controller may be connected to a computer screen 51, which may be equipped with a user interface (UI). This controller C can provide various functions, such as synchronizing actions, providing setpoints, processing signals, performing calculations, and displaying messages / information on a display device (not depicted). It should be understood that the controller C (schematically depicted) may be (partially) inside or outside the housing E, and may have an integrated or modular structure as needed. Those skilled in the art will understand that the interior of the housing E need not be maintained under a strict vacuum; for example, in so-called “ambient TEM / STEM,” a background atmosphere of a given gas is intentionally introduced / maintained within the housing E. Those skilled in the art will also understand that, in practice, it may be advantageous to limit the volume of the housing E such that, where possible, it is substantially close to the axis B' in the form of a small tube (e.g., about 1 cm in diameter) through which the electron beam is passed, but widened to accommodate structures such as the source 4, the sample holder H, the screen 26, the camera 30, the recorder 32, the spectroscopic device 34, etc.

[0048] As defined in this paper, a transmission charged particle microscope M includes: - Charged particle beam source 4, used to emit charged particle beam B; - Sample holder H, used to hold sample S; - Irradiator 6, used to guide the charged particle beam B emitted from charged particle beam source 4 onto sample S; and - Control unit C, used to control the operation of the transmission charged particle microscope M.

[0049] The transmission charged particle microscope M, as defined herein, is arranged to generate and record first and second energy filter fluxes transmitted through the sample of charged particles, as will be discussed in more detail below. The transmission charged particle microscope M, as defined herein, may include a spectroscopic device 34 arranged to generate the first and second energy filter fluxes of the charged particles.

[0050] Turn now Figure 2 The details of this spectroscopic device 34 are shown. Figure 2 It shows Figure 1 An enlarged and more detailed view of an embodiment of the spectral device 34. Figure 2 In the diagram, the electron flux 1 (which has passed through sample S and imaging system 24) is shown propagating along the electron optical axis B'. This flux 1 enters the dispersive device 3 (“electron prism”), where it disperses (fans out) into an energy-decomposing (energy-differentiating) array 5 of spectral sub-beams distributed along the dispersive direction; for illustrative purposes, three of these sub-beams are shown in... Figure 2The numbers are marked as 5a, 5b, and 5c.

[0051] Downstream of the dispersive device 3, the sub-beam array 5 encounters the post-dispersive electron optics 9, where it is magnified / focused, and ultimately guided / projected onto the detector 11 to image the charged particles transmitted through the sample S by the energy-filtered flux. This type of imaging can be accomplished by adjusting the optics 9 so that the image in the plane at the entrance of the spectroscopic device is imaged onto the detector 11. This is energy-filtered transmission electron microscopy (EFTEM). Alternatively, this imaging can be accomplished more indirectly by adjusting the optics 9 so that the image of the dispersive (fan-out) spectral sub-beams is imaged onto the detector, thus forming an electron energy loss spectrum (EELS) of the interaction of the electron beam in the sample. By scanning the focused beam across the sample and recording the EELS spectrum at each location, the energy-filtered image can be reconstructed from the recorded posterior spectrum. This alternative method is called scanning transmission electron microscopy (STEM) EELS. However, the inventors found the EFTEM mode easier to use because it does not require careful adjustment and focusing of the illumination beam. Figure 2 and 3 The various settings in section 4 are applicable to EFTEM mode. It should be noted that detector 11 is known to those skilled in the art, and the microscopy and / or methods defined herein are not, in principle, limited to the use of a specific detector. In EFTEM mode, the spectroscopic device 34 includes a slit for generating charged particles with energy filtering flux. The slit may include one or more slit elements 7, 8 (also referred to as slit edges or slit blades). Figure 2 In the illustrated embodiment, the spectroscopic device 34 includes two slit elements 7 and 8, which can be used to filter a portion of the flux of sub-beams 5a, 5b, and 5c. It is also conceivable to use a single slit element, or more than two slit elements. In the illustrated embodiment, the two slit elements 7 and 8 can be positioned in the first direction d. s Move upwards, where the first direction d s It has at least a component perpendicular to, or substantially perpendicular to, the propagation direction of the sub-beam array 5. The two slit elements 7 and 8 can move together, but in this embodiment, each of the two slit elements 7 and 8 can move independently. Slit elements 7 and 8 allow for precise selection of the sub-beams 5a, 5b, and 5c to be imaged on detector 11. In the method defined herein, first and second energy filtering fluxes of charged particles are generated and recorded. Although slit elements 7 and 8 can be used in this manner, other ways to achieve similar results are contemplated, as will be apparent to those skilled in the art.

[0052] exist Figure 2In the middle, the slit elements 7 and 8 are removed, so that none of the sub-beams 5a-5c are filtered, and an image including all spectral information can be formed on the detector 11 using the optical elements in the post-dispersive electron optics 9.

[0053] Turn now Figure 3 and Figure 4 It demonstrates how to generate and record the first and second energy filter fluxes of charged particles in EFTEM mode.

[0054] exist Figure 3 In this process, slit elements 7 and 8 move in their respective first positions to generate a first energy filtering flux for charged particles. In this case, sub-beam 5a is filtered (i.e., passes through), while other sub-beams 5b and 5c are blocked. Optical elements in the post-dispersive electron optics 9 are used to form an image containing only the spectral information of sub-beam 5a. Thus, the first energy filtering flux for charged particles is generated and recorded.

[0055] exist Figure 4 In this process, slit elements 7 and 8 move to their corresponding second positions to generate a second energy filtering flux for charged particles. In this case, sub-beam 5a is blocked, while other sub-beams 5b and 5c pass through to detector 11.

[0056] As defined in this article, such as Figure 3 As shown, the first energy filtering flux of charged particles consists essentially of unscattered and elastically scattered charged particles. In the illustrated embodiment, the step of generating and recording the first energy filtering flux of charged particles includes so-called zero-loss peak (ZLP) filtering, where the first energy filtering flux of charged particles comprises unscattered and elastically scattered charged particles. This results in an image of sample S with increased contrast.

[0057] As defined in this article, such as Figure 4 As shown, the second energy filtering flux of charged particles consists essentially of charged particles that are inelastically scattered. In the illustrated embodiment, slit elements 7 and 8 are positioned such that they block sub-bundle 5a (ZLP sub-bundle). Therefore, the method may include a step of so-called zero-loss peak (ZLP) blocking. The image obtained with the second energy filtering flux of charged particles can also be used to increase the contrast of the sample. The sample S can be imaged with increased contrast using the first and second recorded energy filtering fluxes. It should be noted that slit element 8 can still be moved within one of the sub-bundles if desired. For example, slit element 8 can be configured to block sub-bundle 5c or other portions of the array of sub-bundles 5.

[0058] The inventors discovered that even for very thin samples (<30 nm), as in the reference... Figure 3The ZLP filtering also improves contrast. This contradicts what is commonly taught. The improvement in SPA contrast achieved through ZLP filtering does not come from removing the blurred background, but from the significant difference in inelastic scattering between the particles and the surrounding ice in which they are embedded.

[0059] The above is demonstrated by the following experimental results. Images of sample S were acquired using an electron microscope M. Ferritin particles embedded in amorphous ice were used as sample S. As those skilled in the art know, microscope M was set to typical SPA acquisition conditions: the sample was cooled to 80 K (to preserve the amorphous structure of the ice and minimize radiation damage), with a diameter of 500 nm and an electron flux of 8 electrons / Å. 2 Parallel illumination is performed at a rate of / second for 5 seconds. The same optical setup is used for each image, but images are captured using charged particles with different energy filtering fluxes obtained by using different positions of slit elements 7 and 8. Figures 2 to 4 The settings and the corresponding results are shown in Figures 5a-5c middle.

[0060] In the first image ( Figure 5a In the image, slits 7 and 8 were not inserted, and the image was created using all electrons in the beam (unscattered, elastic and inelastic). This corresponds to... Figure 2 The setup shown. The deferriprotein particles a (some of which are circled for readability) are difficult to distinguish from the icy region i (one of which is indicated for readability).

[0061] The second image ( Figure 5b In this process, a slit is inserted at the location used for ZLP filtering, allowing only unscattered and elastically scattered electrons (±10 eV) to pass through. This corresponds to... Figure 3 The settings shown. Clearly, ZLP filtering ( Figure 5b This makes ferritin clearly visible. To maximize clarity, the image was processed to remove all spatial frequencies exceeding 1 / 2 nm; however, it should be noted that the unprocessed image also clearly shows that ZLP filtering improves the discernibility of ferritin.

[0062] In the third image ( Figure 5c In this configuration, the upper slit edge 8 is completely retracted, and the lower slit edge 7 moves to the original position of the upper edge. This blocks unscattered and elastically scattered electrons, allowing only inelastic electrons to pass through. This corresponds to... Figure 4 The setup shown. The cold region i and the deferrin particles a are also somewhat distinguishable.

[0063] Figure 5d It shows Figure 5b and 5cThe sum of the images shown is the sum of the ZLP-filtered image and the ZLP-blocked image. As expected, the sum of the ZLP-filtered image and the ZLP-blocked image ( Figure 5d It is very similar to an unfiltered image. Figure 5a This image contains (almost) no contrast between ferritin and ice. Therefore, by adding a ZLP-blocking image to a ZLP-filtered image, ferritin, which is well-identifiable in the ZLP-filtered image, can become unidentifiable. It can be inferred that the ZLP-blocking image must also carry some compelling information.

[0064] Figure 5c This reveals the nature of this information: precisely at the particle's location, the inelastic signal is higher. Logically, this increase in inelastic signal strength is accompanied by a corresponding decrease in elastic signal strength. This decrease in strength (and high-frequency information) makes the particle identifiable in the ZLP-filtered image. Figure 5b ). Figure 6 This schematically illustrates the increase in the intensity of the inelastic signal at deferroferritin particles a, and the corresponding decrease in the intensity of the elastic signal (i.e., unscattered and elastically scattered electrons) at deferroferritin particles a. Therefore, from Figure 6 It can be inferred that both elastic and inelastic signals contain information about the characteristics in sample S, in which case there is particle a in ice layer i.

[0065] EELS spectral analysis revealed that the inelastic signal was primarily affected by plasmon loss, which occurred at energy levels ranging from approximately 10 eV to 40 eV. The inventors... Figure 5c The conclusion is that plasmon interactions in deferroprotein must be stronger than in ice. Plasmons are collective oscillations of valence electrons in the sample. According to Egerton (Egerton, RF, 2011. Electron Energy-Loss Spectroscopy in the Electron Microscope. doi: 10.1007 / 978-1-4419-9583-4), "The fundamental requirement for plasmon excitation is that participating electrons can communicate with each other and share their energy, a condition that is satisfied for delocalized bands but not for atom-like core layers." The inventors hypothesized that plasmons are less likely to be excited in amorphous ice than in granules because the porous and irregular structure of ice makes communication between electrons less efficient than in granules.

[0066] The energy filtering fluxes of the first and second records can be used to image the sample with increased contrast. In view of the foregoing, embodiments of the invention utilize information available in the ZLP-filtered image (i.e., high-frequency information and plasmon contrast) and information available in the ZLP-blocked image (i.e., plasmon contrast) by the following operation: - First, the maximum dose that the sample can withstand before losing structural integrity (typically 40 e / Å at a beam potential of 300 kV). 2 Record the ZLP-filtered image below; and then... - Secondly, at the same location, record ZLP blocking images at similar (or higher) doses.

[0067] Plasmon signals in ZLP-blocked images improve the accuracy of locating particles (or more generally, regions of interest in a sample). This can be achieved in many ways. It is conceivable to process the plasmon image to, for example, locate particles, and use that information to locate particles in the ZLP-filtered image and / or enhance the ZLP-filtered image.

[0068] In another embodiment, the plasmonic image is subtracted from the ZLP-filtered image. Examples of this type of modification are found in... Figure 7 As shown in the image. Here, the image is filtered from ZLP ( Figure 5b Subtract ZLP blocking image from ) Figure 5c This produces high-quality images with greater contrast between the particles and the ice embedded within them. The image can then be digitally processed and / or analyzed to identify the particles. In one embodiment, the particles being imaged and identified can be used to form a three-dimensional (3D) reconstruction of the sample particles. Here, the first and second energy filtering fluxes of the charged particles are used, for example, by subtracting the ZLP blocking image from the ZLP-filtered image.

[0069] Before subtraction, the plasmonic image can be adjusted by a factor. This factor can be optimized for maximum contrast.

[0070] It can be noted that although ZLP-blocking images were recorded for portions of the sample that had already suffered some radiation damage, this is not limiting, as plasmonic signals do not need to (and cannot) carry high-frequency information. Therefore, using the maximum dose to obtain ZLP-filtered images is acceptable.

[0071] It is important to note that the first energy filtering flux can, in principle, include any selected subspectrum of the total spectrum, as long as it contains elastically scattered charged particles. In the embodiment, it is conceivable that the distance and position between slit elements 7 and 8 are selected in a manner that optimizes the image results. The same applies to the distance and position of the slit elements used to generate the second energy filtering flux. In other words, the optimal position of the boundary between the ZLP-filtered image and the ZLP-blocking image can be varied and determined empirically to yield the best image results. This would be helpful if imaging unknown particles or samples. In such cases, it might be helpful to acquire a small subset of images with different slit widths before beginning the actual SPA acquisition.

[0072] Generating and recording charged particles with a second energy flux (e.g., capturing ZLP blocking images) takes additional time, and this increases throughput by a factor of the time required for each stage movement and each exposure. This effect can be offset by the fact that for some samples, 1000 elastic images and 1000 inelastic images yield better reconstruction compared to 2000 elastic images. Additionally, in the embodiments, the beam current can be increased when acquiring inelastic images, which reduces the acquisition time for inelastic images. For small particles, conventional acquisition methods require high-quality thin ice. Grid screening takes a long time to find thin ice (if it exists). Utilizing information from inelastic images, medium or thicker ice can be used, effectively increasing throughput. Finally, it should be noted that when a sample has only a limited number of particles for some reason (say, less than 1000), throughput time is a less relevant parameter; in such cases, it is desirable to obtain all information for each available particle, regardless of throughput time.

[0073] The desired protection is defined by the appended claims.

Claims

1. A method for imaging a sample using a transmission charged particle microscope, the method comprising: - Provide samples; - Provide a beam of charged particles and guide the beam of charged particles toward the sample to generate a certain flux of charged particles that penetrate through the sample; - Generate and record a first energy filtering flux of charged particles that are transmitted through the sample, wherein the first energy filtering flux of charged particles consists essentially of unscattered and elastically scattered charged particles; - Generate and record a second energy filtering flux of charged particles that are transmitted through the sample, wherein the second energy filtering flux of the charged particles consists essentially of charged particles that are inelastically scattered. - Image the sample using the energy filtering flux of the first and second records; - Use the second energy filtering flux of the recorded charged particles to locate the region of interest within the sample; and - Subtract the recorded second energy filtering flux of the charged particles from the recorded first energy filtering flux of the charged particles.

2. The method of claim 1, wherein the step of generating and recording the first energy filtering flux of the charged particles includes zero-loss peak (ZLP) filtering.

3. The method of claim 1 or 2, wherein the step of generating and recording the second energy filtering flux of charged particles includes zero-loss peak (ZLP) blocking.

4. The method of claim 1 or 2, wherein a first dose is used during the step of generating and recording the first energy filtering flux of the charged particles.

5. The method of claim 4, wherein the first dose is equal to the maximum dose that the sample can withstand before losing its structural integrity.

6. The method of claim 5, wherein the maximum dose is 40 e / Å. 2 .

7. The method of claim 4, wherein a second dose is used during the step of generating and recording the second energy filtering flux of the charged particles.

8. The method of claim 7, wherein the step of generating and recording a first energy filtering flux of charged particles is performed first, and the step of generating and recording a second energy filtering flux of charged particles is performed later.

9. The method of claim 8, wherein the second dose is at least equal to the first dose.

10. The method of claim 9, wherein the second dose is at least 40 e / Å. 2 .

11. The method of claim 1, wherein a scaling factor is used on the second energy filtering flux of the recorded charged particles.

12. The method of claim 1 or 2, wherein the sample comprises a plurality of sample particles, and the method includes the step of forming a three-dimensional (3D) reconstruction of the sample particles, wherein the first and second energy filtering fluxes of the charged particles are used.

13. A spectroscopic device comprising: - A dispersive apparatus for receiving a certain flux of charged particles and dispersing the flux of charged particles according to the energy loss of the charged particles; and - Detection system; The characteristic is that the spectroscopic device is arranged for: - Generate and record the first energy filtering flux of charged particles, which consists primarily of unscattered and elastically scattered charged particles; and - Generate and record a second energy filtering flux of charged particles, which consists essentially of charged particles that are inelastically scattered. - Use the second energy filtering flux of the recorded charged particles to locate the region of interest within the sample; and - Subtract the recorded second energy filtering flux of the charged particles from the recorded first energy filtering flux of the charged particles.

14. A transmission charged particle microscope, comprising: - Charged particle beam source, used to emit charged particle beams; - Sample holder, used to hold samples in place; - An irradiator for guiding the charged particle beam emitted from the charged particle beam source onto the sample; and - Control unit for controlling the operation of the transmission charged particle microscope; The characteristic feature is that the transmission charged particle microscope is arranged for: - Generate and record the first energy filtering flux of charged particles, which consists primarily of unscattered and elastically scattered charged particles; and - Generate and record a second energy filtering flux of charged particles, which consists essentially of charged particles that are inelastically scattered. - Use the second energy filtering flux of the recorded charged particles to locate the region of interest within the sample; and - Subtract the recorded second energy filtering flux of the charged particles from the recorded first energy filtering flux of the charged particles.

15. The transmission charged particle microscope of claim 14, comprising the spectroscopic apparatus of claim 13.

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