Method and apparatus for in situ specimen quality inspection in cryogenic focused ion beam milling

Abstract

The present invention relates to a method and dual beam FIB / (S)TEM apparatus for in situ specimen quality inspection in cryogenic focused ion beam milling, comprising the steps of loading a specimen into a specimen holder of the dual beam FIB / (S)TEM apparatus, the (S)TEM apparatus comprising an electron column and a detector, the specimen holder being disposed between the electron column and the detector, using the electron column to direct an electron beam towards the specimen and using the detector to detect electrons passing through the specimen, to obtain an image of the electrons passing through the specimen, and using a scattering pattern in the transmitted electron image to ascertain a measure of specimen thickness and to ascertain whether the image comprises a diffraction signal due to electron diffraction from ice crystals.

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for in situ assessment of the quality of samples for use in transmission electron microscopy (TEM), in particular samples produced by cryogenic focused ion beam milling, to be examined by cryo-electron tomography. [Background technology]

[0002] Cryo-electron tomography (ECT) is a unique technique for structural biology and its applications in, for example, pharmaceutical research, because it allows for the exploration of biological macromolecular structures at near-atomic resolution. Information at this level is important for determining how macromolecules, such as proteins and viruses, interact during health and disease, and how we can intervene in these interactions to identify strategies for treating disease. Because samples for ECT are kept at extremely low temperatures during imaging, the native biological state can be preserved if the sample is rapidly cooled (or fixed) into an amorphous, vitrified state.

[0003] A challenge arises from the requirement that samples for ECT be very thin (<1 μm). This is because contrast in ECT is derived from phase differences in the outgoing waves of elastically scattered electrons induced by changes in sample composition. Inelastic electron scattering must be avoided, which means that the sample typically needs to be thinner than the mean free path for inelastic scattering. In practice, sample thicknesses between 100 and 200 nm are preferred, with thinner often being better. An additional reason for aiming for thinner samples is that lower electron beam energy can be used for thinner samples, reducing sample damage and thereby enabling more information to be extracted from the sample at higher resolution. Relevant biological materials, however, are often not thin enough. Cryogenic focused ion beam (FIB) milling offers a solution to this problem, as the focused ion beam allows material to be milled away with high spatial precision (approximately 10 nm) without affecting unexposed material. In this way, thin slices or lamellae can be cut from cryo-fixed biological material, and then (parts of) these lamellae can be imaged in a TEM to obtain three-dimensional reconstructions at near atomic resolution using ECT.

[0004] For example, US2010 / 0116977 describes a method for TEM sample preparation. The use of a scanning electron microscope (SEM) or scanning transmission electron microscope (STEM) detector in a dual-beam FIB / SEM allows the sample to be thinned using the FIB while the SEM signal is used to monitor the sample thickness. A preferred embodiment can measure the thickness of a STEM sample or prepare a STEM sample by using a precise endpoint detection method that is reproducible and suitable for automatic "endpointing" during lamella preparation. It also provides the user with direct feedback on the sample thickness during manual thinning.

[0005] In addition to the sample thickness, it is also desirable to have a region of interest inside the lamella. A problem with cryo-FIB milling of biological materials is that it is a "blind" process, meaning that the biological material does not provide contrast for the FIB or SEM to reveal differences in composition. For example, it can be very difficult to mill the lamella at the exact correct location and depth in the target material so that a specific biological structure, macromolecular complex, or virus is contained within the lamella. Additionally, it is impossible to assess whether the sample is vitrified at the milling location, which is important because ice formation can cause electron scattering and redistribute or affect the integrity of the biological material. Finally, as explained above, it is difficult to precisely determine the thickness of the lamella during milling, which can be key to achieving the best results.

[0006] A potential solution to the first problem (finding the region of interest for FIB milling) can be found by correlating FIB milling with previously acquired fluorescence microscopy (FM) data obtained from the same cryo-fixed sample. To improve throughput and prevent sample contamination or (partial) devitrification during sample transfer, FM can also be integrated into a FIB-SEM to enable inspection in a vacuum, as disclosed, for example, in WO 2022 / 190136. To enable highly accurate milling of structures within single cells or organelles, FM and cryo-FIB-SEM can even be combined so that all three beams (photons, ions, and electrons) are coincident. In this configuration, FM and SEM can be performed with the sample in the correct position for FIB milling. Summary of the Invention

[0007] A drawback of known methods and devices is that they relate to only one aspect of creating thin lamellae. The inventors have recognized that the quality of a lamella for use in ECT lies in a combination of sample properties. In the case of biological samples, particularly those intended for ECT analysis, the amorphous, vitrified state should be maintained at least until the ECT analysis is complete.

[0008] The object of the present invention is to provide a solution to the above drawbacks by assessing the quality of lamellae for ECT at least with regard to lamella thickness and incomplete vitrification.

[0009] According to a first aspect, the present invention relates to a method for in situ specimen quality inspection in a dual beam cryogenic focused ion beam (FIB) and cryogenic focused ion beam milling in a TEM (Transmission Electron Microscope) or STEM (Scanning Transmission Electron Microscope) ((S)TEM device), the method comprising: loading a sample into a sample holder of a dual beam FIB / (S)TEM instrument, wherein the (S)TEM instrument comprises an electron column and a detector, and the sample holder is positioned between the electron column and the detector; obtaining an image of the transmitted electrons using an electron column to direct the electron beam towards the sample and using a detector to detect the electrons that have passed through the sample; using the scattering pattern in the transmitted electron image to ascertain a measure of the thickness of the sample and to ascertain whether the image comprises a diffraction signal due to electron diffraction; Equipped with.

[0010] The scattering of transmitted electrons is highly dependent on the thickness of the sample, and based on this fact, several methods can be used to determine the thickness of the sample, in particular the lamella, as will be explained in more detail in various embodiments below.

[0011] As noted above, the native biological state can be preserved if the sample is rapidly cooled (or fixed) to an amorphous vitrified state. Samples for ECT are maintained at cryogenic temperatures during sample handling, lamella creation, and sample imaging using ECT. Preserving the native state is crucial for the interpretation and use of ECT results. When the amorphous vitrified state is not preserved—for example, when the amorphous vitrified state in the sample is at least locally altered by the formation of ice crystals—this may indicate that the native state is also not preserved. In addition, this results in additional changes in the image due to electron diffraction at the ice crystals, which can worsen subsequent ECT results. Therefore, potential changes in the image due to electron diffraction at the ice crystals are used to assess whether the sample is still in the desired amorphous vitrified state. When images contain additional diffraction patterns due to electron diffraction at the ice crystals, the native state is likely no longer present in a particular sample, and such samples may be considered low-quality and discarded for use in ECT.

[0012] It should be noted that the method is performed in a dual-beam FIB / (S)TEM, which allows the sample to be thinned using the FIB, while the (S)TEM is used to monitor the sample thickness and image changes due to diffraction from ice crystals. Therefore, the method allows for in situ sample quality inspection. Preferably, the electron beam of the (S)TEM and the ion beam of the FIB can be operated simultaneously or intermittently.

[0013] It is further noted that the term "(S)TEM" as used herein refers to either TEM (transmission electron microscope) or STEM (scanning transmission electron microscope).

[0014] In embodiments, the diffraction pattern is due to electron diffraction from ice crystals, and the diffraction pattern is further evaluated to determine whether cubic and / or hexagonal ice crystals are present in the sample. Because the crystalline structures of cubic ice crystals and hexagonal ice crystals are different, the diffraction patterns produced by these different crystalline forms are also different, which, when present, allows for different crystalline forms to be distinguished from one another. The presence of one or more forms of crystalline ice in a sample can provide clues about the reason for incomplete vitrification. For example, the presence of hexagonal ice can be an indicator of improper and slow kinetics during the freezing of a sample.

[0015] A first embodiment for determining the thickness of a sample, particularly a lamella, uses electron loss due to the interaction between the electron beam and the sample to determine a measure of the sample's thickness. The thicker the sample, the greater the electron loss of the electron beam transmitted through the sample. Because there is a monotonic relationship between sample thickness and electron loss, the electron signal of the transmitted beam can provide a measure of the sample's thickness: the higher the electron signal from the detector, the thinner the sample.

[0016] In embodiments, electron loss is determined by comparing the intensity of the electron beam transmitted through the sample with the intensity of the electron beam when no sample is present, and / or one or more standard samples of known thickness are used to obtain a relationship between the amount of electron loss and sample thickness.

[0017] In an embodiment, the method is used for samples having a thickness in the range of 0 to 100 nm.

[0018] A second embodiment for determining the thickness of a sample, particularly a lamella, uses analysis of the scattering pattern to determine a measure of the most likely scattering angle, which is then used to ascertain the thickness of the sample. When the intensity of the scattered electron beam is determined as a function of scattering angle, an intensity peak is found at the most likely scattering angle. This most likely scattering angle shifts to higher angles for thicker samples. Because this second method determines only the scattering angle at which the intensity peak is located, it is independent of the electron beam intensity and does not require prior sensor calibration.

[0019] In an embodiment, one or more standard samples of known thickness are used to obtain the most likely relationship between scattering angle and sample thickness.

[0020] In an alternative embodiment, the measured most likely scattering angle is compared to the results of a Monte Carlo simulation of electron scattering, preferably as stored in a look-up table, to provide a relationship between the most likely scattering angle and the thickness of the sample.

[0021] In an embodiment, the method is used for samples having a thickness in the range of 75 to 500 nm.

[0022] A third embodiment for determining the thickness of a sample, particularly a lamella, uses analysis of the scattering pattern to obtain a signal for unscattered electrons to provide a bright-field signal and a signal for scattered electrons to provide a dark-field signal, and the ratio between the bright-field and dark-field signals is used to ascertain a measure of the sample's thickness. The advantage of using this relative ratio is that absolute signal levels are not required. Note that the bright-field signal has a monotonic relationship with sample thickness (similar to the first embodiment for determining sample thickness, discussed above). However, the dark-field signal does not have a monotonic relationship with sample thickness; while the dark-field signal increases as the sample becomes thinner, for very thin samples there is less and less material available to scatter electrons, and for such very thin samples, the dark-field signal will decrease with sample thickness. Therefore, the ratio (bright-field signal) / (dark-field signal) increases with decreasing thickness for very thin samples.

[0023] In an embodiment, one or more standard samples of known thickness are used to obtain the relationship between the ratio of (bright field signal) / (dark field signal) and sample thickness.

[0024] In an embodiment, the method is used for samples having a thickness in the range of 50 to 700 nm.

[0025] In an embodiment, the dual beam FIB / (S)TEM instrument uses the following method for determining the thickness of a sample: i. using electron losses due to interaction between the electron beam and the sample according to the first embodiment as described above; ii. using the most probable scattering angle as described in the second embodiment as explained above; iii. Using the ratio between the bright field signal and the dark field signal according to the third embodiment as described above wherein the method is configured to use at least two of the methods for determining the thickness of the sample during the production of the lamella, and the method comprises switching between at least two of the methods for determining the thickness of the sample during the production of the lamella.

[0026] Since each of the methods has the most accurate thickness regime, this embodiment allows for switching between methods for determining the thickness of the sample to use the most accurate method.

[0027] It should be noted that all three methods i, ii, and iii of the embodiment use signals from electrons that have passed through the sample. In the embodiment, the same detector is used to detect electrons transmitted through the sample in at least two of the methods i, ii, and iii for determining the sample thickness, preferably in all three methods i, ii, and iii for determining the sample thickness, and switching between at least two of the methods i, ii, and iii for determining the sample thickness is provided by switching between different methods for analyzing measurements from the detector. Therefore, switching between the different methods i, ii, and iii for determining the sample thickness is actually performed in the controller and / or data analysis device of the dual-beam FIB / (S)TEM apparatus. This allows for easy and / or automatic switching between at least two of the methods i, ii, and iii for determining the sample thickness.

[0028] In an embodiment, the electron energy of the electron beam from the electron column is 30 keV. However, preferably, the energy of the electron beam from the electron column is optimized for thickness determination within a specific thickness range. The inventors have found that the accuracy at a specific thickness may depend on the electron energy.

[0029] Essentially, the thickness determination method as described above is a spot measurement. However, when the spots consist of a matrix of measurement points, they can easily be converted into a map. This allows the thickness determination method to be performed for an area on the lamella being prepared. The thickness determination method can be performed even at locations on the sample where no region of interest is present, thereby preventing unwanted electron beam damage to the region of interest. This can minimize the risk of beam damage and sample devitrification in at least the region of interest.

[0030] In an embodiment, the method is performed on multiple locations on the sample to obtain a measure of the thickness uniformity of the sample or lamella.

[0031] In an embodiment, the detector comprises a scintillator and an optical detector, the scintillator being positioned spaced apart from the sample on the sample holder and between the sample holder and the optical detector, and the method further comprising: converting electrons that pass through the sample into photons using a scintillator; projecting and / or imaging the photons from the scintillator onto an optical detector; Equipped with.

[0032] Therefore, the transmitted electrons and the scattering / diffraction pattern are converted into a corresponding distribution of photons, which is imaged onto an optical detector, such as a CCD image detector, so that the light image as obtained by the optical detector also comprises the scattering / diffraction pattern that was originally present in the transmitted electrons.

[0033] In an embodiment, the dual beam FIB / (S)TEM instrument includes a light-optical microscope that is used to collect photons from the scintillator and image them onto an optical detector. By providing an integrated light-optical microscope, the light-optical microscope can also be used to provide a light-based estimate of the lamella thickness, for example, by using the reflection patterns from the front and back surfaces of the lamella. Preferably, a first thickness of the sample is estimated using a light-optical measurement to prevent exposure of the sample to the electron beam. When the expected thickness is reached, a more accurate thickness determination and / or evaluation of the amorphous state can be performed using (S)TEM detection methods as described above.

[0034] In an embodiment, the scintillator is positioned relative to the sample in the sample holder such that both the sample and the scintillator are within the focal range of the objective lens of the optical system that collects light and images it onto the optical detector. This allows the sample to be examined with a light-optical microscope on the one hand, and an image of the scintillator to be obtained on the other hand. In an embodiment, the sample holder is configured to provide a distance of 300 micrometers between the scintillator and the sample.

[0035] According to a second aspect, the present invention relates to a dual beam FIB / (S)TEM apparatus for micromachining a specimen, the dual beam FIB / (S)TEM apparatus comprising: a sample holder for holding a sample; a FIB unit for projecting a focused ion beam onto a sample held by a sample holder for micromachining the sample; a (S)TEM unit comprising an electron column and a detector, wherein the sample holder is disposed between the electron column and the detector for detecting electrons from the electron column that have passed through the sample; a controller configured, in use, to control the dual beam FIB / (S)TEM apparatus to carry out the steps of the method or embodiments thereof as described above; The integrated combination of

[0036] The various aspects and features described and illustrated in this specification may, to the extent possible, be applied individually, and these individual aspects, in particular those aspects and features described in the accompanying dependent claims, may be made the subject of divisional patent applications.

[0037] The invention will now be explained on the basis of exemplary embodiments shown in the accompanying drawings. [Brief explanation of the drawings]

[0038] [Figure 1] 1 shows a schematic diagram of a first exemplary embodiment of an apparatus for carrying out the method of the present invention; [Figure 2] 2 shows a schematic diagram of a second exemplary embodiment of an apparatus for carrying out the method of the present invention; [Figure 3] 1 shows a schematic of the electron-to-light conversion principle used for (S)TEM detection. [Figure 4] 1 shows a schematic diagram of the principle of sample thickness estimation using the most common scattering angles. [Figure 5] 1 shows a schematic representation of the scattering pattern of an electron beam transmitted through a sample in an amorphous, vitrified state. [Figure 6] 1 shows a schematic representation of the scattering / diffraction pattern of an electron beam transmitted through a sample with incomplete vitrification. DETAILED DESCRIPTION OF THE INVENTION

[0039] 1 shows a schematic diagram of a first exemplary embodiment of an apparatus for carrying out the method of the present invention. The apparatus comprises at least a combination of a focused ion beam (FIB) unit 8 and a (scanning) transmission electron microscope (S)TEM 7, 2, 4. The FIB 8 is configured to focus an ion beam 11 onto a sample 5 on a sample holder 10, and the (S)TEM is configured to focus an electron beam 9 onto the sample 5 on the sample holder 10.

[0040] The (S)TEM comprises an electron column 7 for emitting a primary electron beam 9 and directing said primary beam to a sample 5 supported by a substrate 6 (e.g., a TEM mesh grid) contained in a sample holder 10. The (S)TEM comprises electron optics 2 for projecting electrons 12 transmitted through the sample 5 onto a detector 4. Both the FIB and the (S)TEM are located substantially inside a vacuum chamber 13.

[0041] The inspection device 1 comprises a sample holder 10 for holding a sample 5. The sample holder 10 comprises a cooling system 16 configured to cool the sample 5. Cooling systems per se are known in the art, for example from WO 2020 / 190136 A1. However, other cooling systems may also be applied to maintain the amorphous vitrified state of the sample 5.

[0042] The apparatus of Figure 1 is provided with a controller 15 in the form of a computer, for example a personal computer, said controller 43 being provided with dedicated software configured to carry out one or more methods of the invention or embodiments thereof as described in the description above.

[0043] 1, the sample holder includes a stage for moving the sample 5 relative to the FIB 8 and / or (S)TEM 7, 2, 4. Preferably, the stage is configured to provide six degrees of freedom for moving the sample 5, thereby providing translational movement along the X, Y, and Z axes, and rotational movement about the X, Y, and Z axes.

[0044] Figure 2 shows a schematic representation of a second exemplary embodiment of an apparatus for carrying out the method of the present invention. The apparatus 20 of Figure 2 comprises a scanning electron microscope (SEM) 27 with a vacuum chamber 23 connected to a vacuum pump via a connector 35. Inside the vacuum chamber 23, a sample 40 is placed, which can be irradiated with an electron beam 29. The apparatus 20 comprises an FIB unit 28 for generating, directing, and focusing an ion beam 31 onto the sample 40.

[0045] As shown schematically, a sheet of scintillation material 30 is positioned on the side of the sample 40 facing away from the SEM 27, the sheet of scintillation material 30 being spaced from the sample by a preferred (but not limited to) distance of 300 micrometers. The sample holder comprises a cooling system 41 configured to cool the sample 40 and the sheet of scintillation material 30. Again, cooling systems per se are known in the art, for example from WO 2020 / 190136 A1. However, other cooling systems may also be applied to ensure that the amorphous, vitrified state of the sample 40 is maintained.

[0046] The sheet of scintillator material 30 comprises, for example, a thin slab of YSO:Ce or LYSO:Ce,Ca in the form of a 400 nm or larger transparent window. Preferably, the sheet of scintillator material 30 is provided with a thin layer of transparent conductive material, preferably indium tin oxide (ITO), to avoid charging of the top surface of the sheet of scintillator material, which would otherwise cause beam deflection and pattern distortion. The sample holder is configured to position the sample 40 between the SEM 27 and the sheet of scintillator material 30.

[0047] Located inside the vacuum chamber 23 below the sheet of scintillation material 30 is a microscope objective 22, which is part of a light-optical microscope system. In this particular example, the other main parts of the light-optical microscope system are located outside the vacuum chamber 23 in an illumination and detection box 24.

[0048] Illumination and detection box 24 may include a light source 21, e.g., a laser or LED. Light 36 emitted from light source 21 is directed out of illumination and detection box 24 via a semi-transparent or dichroic mirror 25 and into vacuum chamber 23 via window 32. This light 37, 38 is coupled into microscope objective 22 via mirror 26 to illuminate sample 40. Although an illumination arrangement can be used to illuminate the sample with light and examine the sample under illumination by the light, it is not necessary for the illumination arrangement to obtain an image using electrons transmitted through sample 40 that are converted to light by sheet 30 of scintillation material.

[0049] Light 37, 38 from the sample 40 is collected by the microscope objective 22, directed via mirror 26 and window 32 towards the illumination and detection box 24 and imaged 39 via a semi-transparent or dichroic mirror 25 onto a camera 33, e.g., a CCD detector.

[0050] 2, a light beam for illuminating and / or imaging a sample 40 enters and exits the vacuum chamber 23 through a window 32, which in this example is located in a door 34 of the vacuum chamber 23. The illumination and detection box 24 of the light-optical microscope system may be located outside the vacuum chamber 23 and mounted on the outside of the door 34. However, the illumination and detection portion of the light-optical microscope system may also be contained entirely inside the vacuum chamber 23, for example, mounted on the bottom of the vacuum chamber 23.

[0051] In this exemplary embodiment, it is advantageous to select sheet of scintillator material 30 that is at least substantially transparent, and preferably, the sheet of scintillator material is substantially transparent to light within a wavelength range within the visible spectrum. Thus, sample 40 can be observed by a light optical microscope through sheet of scintillator material 30. Preferably, sheet of scintillator material 30 is transparent in the excitation and emission bands of fluorescent markers that may be used to locate regions of interest.

[0052] As shown schematically in FIG. 2, the sample holder includes a stage for moving the sample 40 to provide six degrees of freedom in the movement of the sample 40, thus providing translational movement along the X, Y, and Z axes, and rotational movement about the X, Y, and Z axes.

[0053] The apparatus of Figure 2 is provided with a controller 43 in the form of a computer, for example a personal computer, said controller 43 being provided with dedicated software configured to carry out one or more methods of the invention or embodiments thereof as described in the description above.

[0054] Figure 3 shows a more detailed schematic of an example sample holder for use in the apparatus of Figure 2. A sample 40 is positioned at a location where both the primary electron beam 29 and the focused ion beam 31 are directed. Electrons passing through the sample are broadened due to scattering of the electrons within the sample. The expanded beam 44 impinges on a scintillator 30, which is positioned a distance from the sample 40 to allow a desired amount of magnification so that the scattering pattern can be specifically detected.

[0055] 3, the side of the scintillator 30 facing the sample 40 is provided with an ITO layer 42. Light 38 from the scintillator is collected by the microscope objective 22.

[0056] Regarding the method for determining the thickness of the sample, it should be noted that the first embodiment, using the electron loss as a function of the thickness of the sample, is very simple and does not require any further explanation.

[0057] Additionally, with respect to the third embodiment, it is noted that obtaining bright-field and dark-field signals is known in the art, for example, reference is made to WO2010 / 0116977A1, which is incorporated herein by reference.

[0058] Regarding the second embodiment, the principle of the sample thickness estimation method using the angular shift of the most probable scattering angle is presented diagrammatically in FIG. 4. A pole piece 50 is configured to emit a primary electron beam 51 onto a sample 53 positioned at a working distance 52 from the pole piece 50. The sample 53 is positioned on a TEM mesh grid 54. At the aperture of the TEM mesh grid 54, a transmitted portion of the primary electron beam 51 may emerge. The primary electron beam 51 is at least partially scattered by the material of the sample 53, resulting in a scattered beam 55. The scattered beam 55 impinges on a detector 61 having a detector size 59, which is positioned at a pole piece-to-detector distance 57. The graph in FIG. 4 represents a summarized annulus signal 58, which clearly has a peak at position 60 at the scattering angle that constitutes the most probable scattering angle. This peak position 60 shifts to lower angles as the sample thickness decreases.

[0059] To find a measure for the thickness of the sample, the measured most likely scattering angle 60 is preferably compared to the results of a Monte Carlo simulation of electron scattering, as stored in a look-up table, to provide a relationship between the most likely scattering angle 60 and the thickness of the sample 53.

[0060] To confirm whether the amorphous vitrification of the sample is still intact, one can look for evidence of the diffraction pattern in the image.

[0061] Figure 5 shows a schematic of the scattering pattern of an electron beam transmitted through the first sample. Since no diffraction pattern is present in this image, it can be concluded that the first sample is in an amorphous, vitrified state.

[0062] Figure 6 shows a schematic of the scattering pattern of an electron beam transmitted through the second sample. Several point-like features are visible around the central spot of the scattered electrons, which result from the diffraction of electrons from ice crystals in the second sample. Because a diffraction pattern is present in this image, it can be concluded that the second sample has at least locally incomplete vitrification.

[0063] The absence of punctate features in Figure 5 indicates that the sample in Figure 5 is substantially amorphous and most likely remains intact and preserved in its native state.

[0064] It should be understood that the above description is included to illustrate the operation of the preferred embodiments and is not intended to limit the scope of the invention. From the above discussion, many variations will become apparent to those skilled in the art that would still be encompassed by the scope of the invention.

Claims

1. A method for quality inspection of a sample in situ during cryogenic focused ion beam milling in a dual-beam FIB / (S)TEM apparatus, wherein the method is: The steps include loading the sample into the sample holder of the dual-beam FIB / (S)TEM apparatus, wherein the (S)TEM apparatus comprises an electron tube and a detector, and the sample holder is positioned between the electron tube and the detector. The steps include using the electron microscope to direct an electron beam toward the sample, and using the detector to detect electrons passing through the sample, thereby obtaining an image of the electrons that have passed through the sample. The steps include: confirming the thickness of the sample and using the scattering pattern of transmitted electrons in the image to confirm whether the image contains diffraction signals due to electron diffraction; A method that includes [a certain feature].

2. The method according to claim 1, wherein the electron loss resulting from the interaction between the electron beam and the sample is used to determine a measure of the thickness of the sample.

3. The method according to claim 2, wherein the electron loss is determined by comparing the intensity of the electron beam that has passed through the sample with the intensity of the electron beam when the sample is not present, and / or one or more standard samples of known thickness are used to obtain a relationship between the amount of electron loss and the thickness of the sample.

4. The method according to claim 2, wherein the method is used for a sample having a thickness in the range of 0 to 100 nm.

5. The method according to claim 1, wherein the scattering pattern is analyzed to determine a measure of the most likely scattering angle, and the most likely scattering angle is used to determine a measure of the thickness of the sample.

6. The method according to claim 5, wherein one or more standard samples having known thicknesses are used to obtain the most likely relationship between the scattering angle and the thickness of the sample.

7. The method according to claim 5, wherein the most likely scattering angle measured is compared with the results of a Monte Carlo simulation of electron scattering, and a relationship between the most likely scattering angle and the thickness of the sample is provided.

8. The method according to claim 5, wherein the method is used for a sample having a thickness in the range of 75 to 500 nm.

9. The method according to claim 1, wherein the scattering pattern is analyzed to obtain a signal for non-scattered electrons to provide a bright-field signal and a signal for scattered electrons to provide a dark-field signal, and the ratio between the bright-field signal and the dark-field signal is used to determine a measure of the thickness of the sample.

10. The method according to claim 9, wherein the method is used for a sample having a thickness in the range of 50 to 700 nm.

11. The dual-beam FIB / (S)TEM apparatus uses the following method for determining the thickness of the sample, namely, i. Using the electron loss resulting from the interaction between the electron beam and the sample as described in claim 2, 3, or 4, ii. Using the most likely scattering angle described in claim 5, 6, 7, or 8, iii. Using the ratio between the bright-field signal and the dark-field signal as described in claim 9 or 10. The method according to claim 1, wherein the method is configured to use at least two of the methods, and the method comprises the step of switching at least two of the methods for determining the thickness of the sample during the production of the lamellar.

12. The method according to claim 11, wherein the same detector is used to detect electrons transmitted through the sample in at least two of the methods i, ii, and iii for determining the thickness of the sample, and the step of switching between at least two of the methods i, ii, and iii for determining the thickness of the sample is provided by switching between different methods for analyzing the measurements from the detector.

13. The method according to any one of claims 1 to 10, wherein the method is performed on a plurality of locations on the sample to obtain a measure of thickness uniformity.

14. The method according to claim 11, wherein the method is performed on a plurality of locations on the sample to obtain a measure of thickness uniformity.

15. A dual-beam FIB / (S)TEM apparatus for micromachining a sample, wherein the dual-beam FIB / (S)TEM apparatus is A sample holder for holding the aforementioned sample, A FIB unit for projecting a focused ion beam onto the sample held by the sample holder in order to perform micro-machining of the sample, (S)TEM unit comprising an electron microscope tube and a detector, wherein the sample holder is positioned between the electron microscope tube and the detector to detect electrons that have passed through the sample from the electron microscope tube. A controller configured to control the dual-beam FIB / (S)TEM apparatus to perform the steps of the method according to any one of claims 1 to 10 when in use, A dual-beam FIB / (S)TEM system featuring an integrated combination of these elements.

16. A dual-beam FIB / (S)TEM apparatus for micromachining a sample, wherein the dual-beam FIB / (S)TEM apparatus comprises: A sample holder for holding the aforementioned sample, A FIB unit for projecting a focused ion beam onto the sample held by the sample holder in order to perform micro-machining of the sample, (S)TEM unit comprising an electron microscope tube and a detector, wherein the sample holder is positioned between the electron microscope tube and the detector to detect electrons that have passed through the sample from the electron microscope tube. A controller configured to control the dual-beam FIB / (S)TEM apparatus to perform the steps of the method according to claim 11 when in use, A dual-beam FIB / (S)TEM system featuring an integrated combination of these elements.

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