METHOD, DEVICE AND SYSTEM FOR REDUCING OFF-AXIAL ABERRATION IN ELECTRON MICROSCOPY

Abstract

The invention relates to a method for electron microscopy. The method comprises providing an electron microscope, generating an electron beam and an image beam, adjusting one of the beam and of the beam and the image beam to reduce off-axial aberrations and correcting a diffraction pattern of the resulting modified beam. The invention also relates to a method for reducing throughput time in a sample image acquisition session in transmission electron microscopy. The method comprises providing an electron microscope, generating a beam and an image beam, adjusting one of the two to reduce off-axial aberrations and filtering the resulting modified image beam. The invention further relates to an electron microscope and to a non-transient computer-readable medium with a computer program for carrying out the methods.

Claims

1. A method for electron microscopy comprising providing an electron microscope (1) comprising a sample component (2), at least one beam generator (4), at least one adjusting component (6, 8) and at least one diffraction correcting component (8, 10); securing a sample by using the sample component (2); generating an electron beam (12) by using the beam generator (4); generating an image beam (14) by directing the beam (12) to the sample component (2); adjusting at least one of the beam (12) and the image beam (14) by using the adjusting component (6,8) to obtain at least one modified image beam (16) wherein the adjusting is performed in such a way, that off-axial aberrations inflicted on at least one of the beam (12) and the image beam (14) are minimized; and correcting a diffraction pattern in the modified image beam (16) by using the diffraction correcting component (10).

2. The method according to the preceding embodiment wherein adjusting at least one of the beam (12) and the image beam (14) by the adjusting component comprises at least one of shifting and tilting at least one of the beam (12) and the image beam (14) respectively.

3. The method according to the preceding embodiment wherein both shifting and tilting are applied to at least one of the beam (12) and the image beam (14) and wherein the amount of tilting of at least one of the beam (12) and the image beam (14) depends on the amount of shifting of at least one of the beam (12) and the image beam (14) respectively and wherein the amount of tilting is computed based on at least one of coma and astigmatism introduced as a consequence of the shift.

4. The method according to any of the preceding method embodiments wherein the adjusting comprises at least shifting of up to 3 μm, preferably up to 5 μm, even more preferably up to 10 μm of at least one of the beam (12) and image beam (14).

5. The method according to any of the preceding method embodiments wherein the adjusting comprises shifting and tilting the beam (12) and the image beam (14) and wherein the shift of the image beam (14) is substantially equal and opposite to the shift of the beam (12) and the tilt of the image beam (14) is substantially equal and opposite to the tilt of the beam (12).

6. The method according to any of the preceding method embodiments and with the features of embodiment 2 wherein the diffraction correcting component (10) comprises an additional lens (10) and a double deflector unit (8) and wherein the additional lens (10) is configured to generate a magnified diffraction pattern, and the double deflector unit (8) is configured to de-tilt at least one of the image beam (14) and the modified image beam (16).

7. The method according to any of the preceding embodiments wherein correcting the diffraction pattern in the modified image beam (16) comprises tilting the modified image beam (16) to center the diffraction pattern.

8. The method according to any of the preceding method embodiments used to improve at least one of contrast and resolution in scanning transmission electron microscopy and wherein the adjusting component (6,8) comprises quadrupoles and wherein the electrostatic quadrupoles reduce astigmatism created by shifting at least one of the beam (12) and image beam (14), and the diffraction correcting component (10) comprises scan coils, and the method further comprises using the scan coils to tilt the beam (12) before directing it to the sample component (2) and de-tilting the image beam (14) below the sample component (2).

9. Use of the method according to any of the preceding method embodiments on electron microscopes without inbuilt spherical aberration correction.

10. A method for reducing throughput time in a sample image acquisition session in transmission electron microscopy comprising providing an electron microscope (1) comprising a sample component (2), at least one beam generator (4), at least one adjusting component (6,8), and at least one filtering component (20); securing a sample by using the sample component (2); generating an electron beam (12) by using the beam generator (4); generating an image beam (14) by directing the beam (12) to the sample component (2); adjusting at least one of the beam (12) and the image beam (14) by using the adjusting component (6,8) to obtain at least one modified image beam (16) wherein the adjusting is performed in such a way, that off-axial aberration of the modified image beam (16) is minimized; filtering the modified image beam (16) via the filtering component (20) to reduce resolution-deteriorating effect of chromatic aberration on the modified image beam (16) resulting from the adjusting of the at least one of the beam (12) and the image beam (14).

11. The method according to the preceding embodiment wherein the adjusting is performed repeatedly to obtain a plurality of different modified image beams (16).

12. The method according to any of the preceding method embodiments 10 to 11 wherein the adjusting comprises shifting and tilting the beam (12) and the image beam (14) and wherein the shift of the image beam (14) is substantially equal and opposite to the shift of the beam (12) and the tilt of the image beam (14) is substantially equal and opposite to the tilt of the beam (12).

13. The method according to any of the preceding method embodiments 10 to 12 used to reduce sample throughput time in single particle analysis and wherein adjusting comprises at least one of a beam shift and an image shift as well as at least one of a beam tilt and an image tilt, and “stage shift” refers to physically moving the sample component (2) (12) so that a different part of the sample can be imaged, wherein the throughput time of sample image acquisition is reduced by decreasing the number of stage shifts and increasing the number of at least one of beam and image shifts performed in the sample image acquisition session.

14. The method according to the preceding embodiment wherein the number of at least one of beam shifts and image shifts taken without applying a stage shift comprises at least 10 shifts, preferably at least 20 shifts, such as at least 25 shifts.

15. The method according to any of the preceding embodiments 10 to 14 and with the features of embodiment 13 further comprising increasing the distance of at least one of beam shifts and image shifts and wherein the distance of the at least one of beam shifts and image shifts is increased by at least 2 times, preferably at least 3 times, more preferably at least 5 times as compared to standard single particle analysis shift distance of about 1-2 μm.

16. The method according to any of the preceding embodiments 10 to 15 and wherein adjusting comprises at least one of a beam shift and an image shift as well as at least one of a beam tilt and an image tilt wherein the tilt t applied to the beam (12) is related to the shift s applied to the image beam (14) as t=−K.Math.s/Cs, where K denotes off-axial coma and Cs denotes spherical aberration.

17. The method according to any of the preceding embodiments 10 to 16 wherein the filtering component (20) comprises an electron energy filter and wherein the filter component (20) filters out electrons with an energy loss above 10 eV, preferably above 5 eV, more preferably above 3 eV.

18. An electron microscope (1) comprising a sample component (2) configured to receive a sample; at least one beam generator (4) configured to generate an electron beam (12) wherein the beam (12) is configured to generate an image beam (14) upon being directed to the sample component (2); at least one adjusting component (6,8) configured to adjust at least one of the beam (12) and the image beam (14) to obtain at least one modified image beam (16) wherein the adjusting component is further configured to minimize off-axial aberration inflicted on at least one of the beam (12) and the image beam (14); and at least one diffraction correcting component (10) configured to correct a diffraction pattern in the modified image beam (16) resulting from at least one of beam and image shifts.

19. The electron microscope (1) according to any of the preceding device embodiments, wherein the adjusting component (6, 8) comprises at least a pair of beam deflectors (6) and a pair of image deflectors (8), each pair configured to at least one of shift and tilt the beam (12) and the image beam (14) respectively.

20. The electron microscope (1) according to the preceding embodiment wherein the adjusting component (6,8) is configured to shift and tilt the beam (12) and the image beam (14) and wherein the shift of the image beam (14) is substantially equal and opposite to the shift of the beam (12) and the tilt of the image beam (14) is substantially equal and opposite to the tilt of the beam (12).

21. The electron microscope (1) according to any of the preceding device embodiments wherein the diffraction correcting component (10) comprises an additional lens (10) and a double deflector unit (8).

22. The electron microscope (1) according to any of the preceding device embodiments configured to operate without an inbuilt image corrector introducing a shift-dependent tilt to reduce off-axial coma.

23. A non-transient computer-readable medium with a computer program for carrying out the method for transmission electron microscopy according to any of the preceding method embodiments.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0225] FIGS. 1a and 1b schematically depict the steps of the methods according to two embodiments of the invention;

[0226] FIGS. 2a and 2b depict an embodiment of an electron microscope according to prior art;

[0227] FIG. 3 depicts an embodiment of an electron microscope according to one embodiment of the invention and configured to execute the inventive method;

[0228] FIG. 4 depicts another embodiment of an electron microscope according to the invention and configured to execute the inventive method;

[0229] FIG. 5a depicts a prior art method for single particle analysis in electron microscopy;

[0230] FIG. 5b depicts a method for single particle analysis according to an embodiment of the invention;

[0231] FIG. 6 depicts an exemplary embodiment of a Zemlin tableau used to compute aberration coefficients of the obtained images.

DESCRIPTION OF EMBODIMENTS

[0232] FIG. 1a schematically depicts an embodiment of a method according to the invention. The method can be preferably executed by an electron microscope. The electron microscope can be a transmission or a scanning microscope. The applications of the method can be to various branches of electron microscopy such as TEM, STEM, SPA, Tomography and others.

[0233] The order of the steps as described here, in the claims, and in the rest of the application can be variable, and the method can be applied with the steps performed in a different order.

[0234] In step S1, a sample can be secured via a sample component. That is the sample can be placed on a sample/specimen plate or grid. The sample can also be prepared in a certain way, such as encased in vitrified ice. In single particle analysis, the specimen can be placed on a grid comprising a plurality of holes, each containing a foil of ice with the sample molecule encased in it.

[0235] In step S2, an electron beam can be generated via a beam generator. Various method of providing a beam for electron microscopy are known in the art.

[0236] In step S3, an image beam can be generated by directing the beam to the sample component. That is, in the case of transmission electron microscopy, the beam can pass through the sample secured via the sample component. In the case of scanning electron microscopy, the beam can hit the surface of the sample secured via the sample component.

[0237] In step S4, at least one of the beam and the image beam can be adjusted to obtain at least one modified image beam. That is, either or both of the beam and image beam can be shifted and/or tilted by using electron deflectors. This can result in a horizontally displaced imaging area. The shift can be larger than those previously used in electron microscopy. That is, a beam/image beam shift of up to about 10 pm (as compared to the typical 1-2 μm) can be achieved. The modified image beam can then comprise a different part of the sample as compared to the image beam (the previous image beam). Preferably both a shift and a tilt are used.

[0238] The adjustment can be done is such a way, so as to reduce off-axial aberration in the beam, the image beam and/or the modified image beam. For example, the applied tilt can be dependent on the applied shift so as to maximally reduce off-axial aberration.

[0239] Large image beam shifts can lead to increased off-axial aberration (such as off-axial coma), which then needs to be corrected to obtain an image that is in focus. Off-axial coma can generally be corrected by implementing specialized software that makes tilt corrections to the beam and/or image beam depending on the shift that has been applied on the beam/image beam.

[0240] In step S5, a diffraction pattern in the modified image beam can be corrected. This correction can be done in various ways. In STEM, electrostatic quadrupoles can be used to make scan-dependent stigmator corrections. In Themis microscopes, scan coils below the sample can be used for de-tilting the beam after a large image beam shift, thereby reducing off-axial aberration. In SPA, but also in STEM or TEM, an additional electromagnetic lens with a double deflector unit can be placed between the objective lens and the projector system. The additional lens can be used to “de-tilt” (that is, undo the effects of the previous tilt) the shifts of the diffraction pattern while making coma-free image beam shifts.

[0241] FIG. 1b schematically depicts an embodiment of a slightly different method according to one embodiment of the invention. This method is preferably used in transmission electron microscopy, as opposed to the previous one, which can be used in both TEM and STEM. Steps S1 to S4 of the present method are the same and steps S1 to S4 of the previous method depicted in FIG. 1a and described above.

[0242] In step S5′, the modified image beam is filtered via the filtering component. This is done to avoid blur due to the electrons scattering inelastically on the constituents of the sample. This filtering is not necessary in STEM, but plays a significant role in TEM, as it allows to keep resolution loss due to (off-axial) chromatic aberrations low while applying large shifts to the beam and/or image beam.

[0243] FIGS. 2a and 2b schematically depict an electron microscope 1′ according to the prior art. Both figures depict a sample component 2′ and adjusting component 6′, 8′ (depicted as two pairs of deflectors 6′ and 8′). Beam 12′ is shown being adjusted by the adjusting component 6′, 8′. Image beam 14′ and modified image beam 16′ are also depicted.

[0244] In FIG. 2a, a shift of the beam 12′ is shown. That is, the beam 12′ is deflected by the adjusting component 6′. The image beam 14′ still passes through the optical center of back focal plane 30′.

[0245] In FIG. 2b, a tilt of the beam 12′ is shown. That is, the beam 12′ is tilted by the adjusting component 6′. The image beam 14′ does not pass through the optical center of the back focal plane 30′.

[0246] FIG. 3 depicts an electron microscope 1 according to one embodiment of the invention. The electron microscope 1 can comprise a sample component 2 where a sample or a specimen can be placed and/or secured. The sample component 2 can comprise a plate, a grid with a plurality of holes, or another component typical in electron microscopy.

[0247] Also depicted in the figure is a beam generator 4. The beam generator 4 can provide an electron beam. The beam generator 4 can comprise an electron gun or another typical electron beam generator. Adjusting components 6, 8 are depicted as beam deflection coils and image beam deflection coils. The adjusting component 6, 8 can shift and/or tilt beam 12 and/or image beam 14. A diffraction correcting component 10 is depicted as an additional lens 10 on the present figure. They can be used to shift the image beam back to the optical axis, as well as to de-tilt the image beam. The coils 6 can additionally or alternatively be used to generate a Zemlin tableau which can in turn be used to quantify aberration coefficients and to minimize them by software. Particularly, spherical aberration can be corrected by when the microscopes is equipped with a built-in Cs corrector. However, the diffraction correcting component 10 can also comprise an additional electromagnetic lens 10 configured to create an intermediate image of back-focal plane 32 in order to place a phase plate.

[0248] The beam 12 emitted by the beam generator 4 is referred to as image beam 14 upon passing through the sample component 2. The image beam 14 is further modified to yield a modified image beam 16 (depicted in the present figure after additional adjusting by deflectors 8 and after the back focal plane 32.

[0249] The electron microscope as depicted in FIG. 3 can be preferably used for STEM or TEM analysis. Since the figure is schematic, further pieces of equipment are not shown or numbered (such as further lenses for magnification and/or focusing of the beam, the image beam, the modified image beam and/or further components).

[0250] FIG. 4 depicts another schematic embodiment of an electron microscope 1. Preferably, it can be used for single particle analysis in TEM. As before, the beam generator 4 produces a beam 12. The beam 12 is then adjusted by the adjusting component 6, 8. In the depicted case, beam deflectors 6 apply a shift and a tilt to the beam, resulting in skew illumination. The skewed beam 12 then passes through the sample component 2, resulting in an image beam 14. The image beam 14 is then filtered by a filtering component 20. The filtering component 20 can comprise an adjustable slit that only lets electrons of certain energies pass. For example, electrons with an energy loss of approximately below 3 eV can be allowed to pass, to avoid image blurriness due to inelastic electron scattering. After filtering, the image beam 14 hits the back focal plane 30 off-center. It is then further adjusted by the deflectors 8 to return to the optical center and result in the modified image beam 16. Note, that the filtering component 20 is depicted and described in the present application only schematically. It is described in detail in the applicant's European patent application EP 2 388 796 A1, incorporated herein by reference.

[0251] FIGS. 5a and 5b schematically depicts a sample component 2, preferably used in single particle analysis. The sample component 2 can comprise a plurality of holes or apertures 22. The holes 22 can house a sample or specimen encased in ice foils.

[0252] FIG. 5a shows a previously known way of obtaining a plurality of sample images, that is, the prior art. Image acquisitions 42, shown as smaller circles in the holes 22 depict the area imaged by a particular beam/image orientation. Small pale arrows correspond to image beam shifts 44, typically performed on a small scale within one hole in the prior art. The image beam shifts 44 are highlighted in black in one of the holes to make them easier to locate. Longer arrows that connect different holes 22 correspond to stage moves 46. The stage moves 46 are typically used in the prior art to obtain images of a different hole 22 of the sample grid. The stage moves 46 typically take about 20-60 seconds to perform, leading to undesirably increased acquisition times for single particle analysis. Image beam shifts, on the other hand, take about 1 second to perform.

[0253] In FIG. 5a, three images acquired with an electron microscope take about 1 stage movement (about 30 seconds), 3 image beam shifts (about 3*1 seconds) and 3 exposures (about 3*4 seconds). Therefore, the average acquisition time per image is about 15 seconds.

[0254] FIG. 5b depicts the presently disclosed inventive way of performing single particle analysis. Image beam shifts 44, now depicted by longer pale arrows can be used to image all or a plurality of neighboring holes 22 starting from a central one. This can be achieved by enabling larger image beam shifts which were previously not possible for single particle analysis. Advantageously, the number of stage moves 46 can be significantly reduced. This can lead to a much improved acquisition time in single particle analysis. The method of FIG. 3b is also preferably performed with dynamic correction of coma, astigmatism and defocus, as well as energy filtering (for some cases, for example SPA).

[0255] In FIG. 5b, acquiring 27 images takes approximately 1 stage movement (about 30 seconds), 27 image beam shifts (about 27*1 second), 27 exposures (about 27*4 seconds). This yields an average time of 6 seconds per image, significantly increasing the throughput time of a sample.

[0256] FIG. 6 depicts an exemplary embodiment of a Zemlin tableau. The Zemlin tableau comprises a plurality of subgraphs 50 which are each Fourier transforms of different TEM images taken by tilting the incident beam around the optical axis through a series of azimuthal angles. As depicted in the figure, the positions at which each of the subgraphs 50 are plotted in the Zemlin tableau 5 correspond to the tilt applied to the incident beam used to record each of the TEM images of which each of the subgraph shows the Fourier transform. That is, the central subgraph corresponds to an image taken without any tilt, while the top one corresponds to an image taken with a tilt in this direction with respect to the central image and when looking from above. From the different subgraphs 50, aberration coefficients can be calculated and then implemented in correcting software. This is further explained above when discussing the Zemlin tableau. The different tilts result in different first order aberrations, which can be analyzed with specialized software to derive all aberrations (and not just first order ones), including the on-axial coma.

[0257] Whenever a relative term, such as “about”, “substantially” or “approximately” is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., “substantially straight” should be construed to also include “(exactly) straight”.

[0258] Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be the preferred order, but it may not be mandatory to carry out the steps in the recited order. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may not be mandatory. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Y1), . . . , followed by step (Z). Corresponding considerations apply when terms like “after” or “before” are used.