WO2010125347A2

WO2010125347A2 - Inelastic scanning confocal electron microscopy

Info

Publication number: WO2010125347A2
Application number: PCT/GB2010/000852
Authority: WO
Inventors: Peter David Nellist; Peng Wang; Angus Ian Kirkland; Gavin Joseph Behan
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2009-05-01
Filing date: 2010-04-28
Publication date: 2010-11-04
Anticipated expiration: 2011-11-01
Also published as: WO2010125347A3; GB0907600D0

Abstract

To perform detection of electrons scattered inelastically from a specimen, a confocal electron microscope is configured so that the post-specimen lens system in respect of electrons of a predetermined energy loss below the incidence energy is confocal with the pre-specimen lens system in respect of electrons of the incidence energy. This avoids the need to correct for chromatic aberration and instead takes advantage of the chromatic aberration of the post-specimen lens system to selectively detect electrons experiencing the energy loss of interest without the need to use a costly energy filter. The confocal configuration is conveniently achieved by first configuring the confocal electron microscope to achieve a confocal configuration with electrons of a first incidence energy and then changing the incidence energy by an energy offset selected to provide a confocal arrangement for the energy loss of interest.

Description

Inelastic Scanning Confocal Electron Microscopy The present invention relates to confocal electron microscopy.

Scanning confocal electron microscopy (SCEM) is an imaging mode in electron microscopy. Use of a confocal arrangement in an electron microscope provides improved depth resolution and selectivity over optical sectioning in the scanning transmission electron microscopy (STEM) mode.

Confocal scanning optical microscopy (CSOM) has become a successful and widespread technique in light optics. Its strength lies in its strong depth discrimination property. Scattering from points away from the confocal point, both laterally and parallel to the beam, is rejected and detected less strongly than the in-focus scattering. In a conventional optical system where, the focusing optics act only on either the scattered radiation, as in a conventional microscope, or on the incident beam to form a focused beam scanning microscope, the out-of-focus scattering is still present in the image, forming a blurred intensity background and confusing image interpretation. Confocal microscopy provides a depth discrimination property, suppressing the contribution of the out-of-focus scattering, and allowing probing of specific depths in a specimen or recordal of a stack of ID images with a transverse section at different depths in thick specimens to form a 3D image. The confocal configuration leads to improved resolution in direction perpendicular to the optics axis, as compared to a scanning optical microscope.

In electron microscopy, a confocal configuration in principle provides analogous benefits over STEM. However, SCEM is in practice limited by the spherical aberration inherent in electron lenses that reduces the theoretically achievable resolution. SCEM has however been demonstrated. Frigo et al., Applied Physics Letters 81, 2112 (2002), and a related patent US-6,548,810, disclose improved image contrast in very thick specimens using SCEM that showed images of buried structure at micrometre lateral resolution. The longitudinal resolution in the optical sectioning was strongly limited by small apertures (subtending less than 10 mrad semi-angle) they used due to large spherical aberration that is inherent in conventional round-lens electron optics. Whereas the lateral resolution of an optical system is inversely proportional to its numerical aperture, the depth of focus varies in inverse proportion to the square of the numerical aperture.

The advent of spherical aberration correctors for both TEM and scanning transmission electron microscopy (STEM), as disclosed in Nellist et al., Science 305, 1741 (2004), has dramatically improved the spatial resolution in 2D images because the correction of spherical aberration allows a greater numerical aperture to be used in the lenses. In addition to increasing the lateral resolution, a larger convergence angle also leads to a reduced longitudinal depth of field, which is likely to approach a few nanometres with the latest aberration-corrected microscopes.

Having such a narrow depth of field offers the possibility of depth sectioning of specimens at nanometre resolution, and the determination of the height of single atoms has been demonstrated using an aberration corrected STEM instrument. Recent work has shown that the depth resolution of an aberration corrected STEM system rapidly deteriorates when laterally extended systems are imaged because of their out of focus contribution to the image. This can be explained in terms of a so-called "missing cone" in the three-dimension optical transfer function that has long been known in light optics. In the STEM case, the depth resolution of an object with characteristic lateral length scale, d , can be expressed as d/a. Because the aperture α in an electron microscope is very small (around 30 mrad even in a current aberration-corrected instrument), the depth resolution of a 5 nm nanoparticle as an example is around 170 nm, much worse than the FWHM (full width half maximum) depth of the probe intensity about 5nm at 200keV. However, working in a confocal mode fills this missing cone in the transfer function, making depth discrimination of laterally extended objects possible at maximum longitudinal resolution.

Nellist et al., Applied Physics Letters 89, 124105 (2006) demonstrates that spherical aberration corrected TEM/STEM instruments are capable of working in this confocal mode and predicts that the depth resolution is about 5.2 nm on a 300keV instrument with a 30mrad semi-angle aperture. This holds out the possibility of useful 3D nanometre-scale resolution by optical sectioning in SCEM, an entirely new form of electron microscopy.

In principle, SCEM can be operated with two imaging modes, namely elastic SCEM or inelastic SCEM differentiated by detecting either elastically scattered electrons or inelastically scattered electrons that have sustained a particular energy loss in the specimen.

Cosgriff et al., Ultramicroscopy 108, 1558 (2008) and D'Alfonso et al., Ultramicroscopy 108, 1567 (2008) disclose theoretical examination of the 3D imaging performances in these two systems on the identification and location of isolated impurities. This work shows elastic SCEM does not transfer first-order contrast so that image contrast is weak. However, inelastic SCEM gives improved contrast and depth resolution. Inelastic SCEM also provides atomic species selectivity because the energy loss of inelastically scattered electrons is characteristic of the species with which the scattering interaction occurs. Thus, inelastic SCEM in principle allows reconstruction of 3D distributions of specific elements on a nanometre scale from a series of 2D inelastic SCEM optical sections. This technique is analogous to confocal fluorescence microscopy in light microscopes.

To implement inelastic SCEM, an energy filter is used, this leading to the alternative name of energy filtered SCEM (EFSCEM). The energy filter is arranged between the specimen and the detector and is used to selectively detect electrons of a given energy (or energies). With knowledge of the incidence energy of electrons incident on the sample, this equates to detection of inelastically scattered electrons that have experienced a given energy loss. However, an energy filter is a complex and expensive piece of equipment. Typically an energy filter might cost something of the order of 10- 30% of the cost of the rest of the scanning confocal microscope.

Another perceived problem to the implementation of inelastic SCEM in a confocal electron microscope is the residual uncorrected chromatic aberration C, which changes the focal length of the post-specimen lens system by an amount Az

A* = AE_loss £- (1) for the inelastic scattering of electrons incident with an incidence energy E₀ and experiencing an energy loss AE_loss . Furthermore, typical energy filtered electron microscope images are recorded using electrons with energy losses that range over a window ΔE_window of several eV to ensure a high enough signal. With a typical values of C=I .5 mm, ΔE_window = 1 OeV , and incidence energy £o=200keV, the resulting focal spread will be 75nm, much greater than the 5 run depth of focus we might expect in an inelastic SCΕM experiment. It is therefore perceived that this chromatic aberration would need to be corrected to implement inelastic SCΕM. However, this is practically difficult to achieve. Although spherical aberration correctors are becoming widespread because of their commercial availability, there is currently only one instrument fitted with a prototype chromatic aberration corrector because it is technologically much harder. The chromatic aberration corrector is also expensive.

Therefore, whilst inelastic SCΕM is in principle desirable, practical implementation suffers from these practical difficulties and costs.

According to a first aspect of the present invention there is provided a method of operating a confocal electron microscope to detect electrons scattered inelastically from a specimen with an energy loss of interest, the confocal electron microscope comprising: an electron source for emitting electrons with an incidence energy; a pre-specimen lens system for focussing the electrons emitted from the electron source to a point in a specimen; an electron detector for detecting electrons incident at a localised region; and a post-specimen lens system for focussing electrons scattered from the point onto the localised region, wherein the post-specimen lens system has chromatic aberration, the method comprising configuring the confocal electron microscope so that the post- specimen lens system in respect of electrons of the energy loss of interest below the incidence energy is confocal with the pre-specimen lens system in respect of electrons of the incidence energy.

According to a second aspect of the invention, there is provided a control apparatus for controlling a confocal electron microscope to perform a similar method.

Therefore, the present invention allows the performance of inelastic SCΕM. However, this is done with a post-specimen lens system that has chromatic aberration, rather than correcting for such chromatic aberration, for example using a chromatic aberration as part of the post-specimen lens system. This is in contrast to the perception that inelastic SCΕM would require correction of chromatic aberration to reduce focal spread. Rather, it has been appreciated that the chromatic aberration does not deteriorate the depth resolution of SCΕM, but actually can provide energy selectivity in a similar manner to an energy filter. In particular advantage is taken of the fact that the focal depth of the post-specimen lens system varies with the energy of the electrons passing therethrough. Thus, the confocal electron microscope is configured so that the post-specimen lens system in respect of electrons of the energy loss of interest below the incidence energy is confocal with the pre-specimen lens system in respect of electrons of the incidence energy. This has the result that the detector selectively detects electrons experiencing the energy loss of interest. Electrons having other energies are rejected, this energy discrimination being achieved based on substantially the same effect as that by which a confocal configuration provides depth resolution.

Thus, as well as avoiding the need for correction of chromatic aberration, it is possible to provide energy selection allowing the performance of inelastic SCEM without the need for an energy filter and thereby providing a significant saving on the complexity of the overall microscope

The efficacy of the chromatic aberration in providing energy selectivity is demonstrated by experiments and theoretical calculations set out below. However, the observation can be relatively simply explained, as follows. Electrons of the energy loss of interest for which the post-specimen lens system has been configured will be correctly focussed at the localised region detected by the detector. Because of the chromatic aberration of the post-specimen lens system, electrons of any other energy loss will only be focused at the localised region if they arise from scattering above or below the confocal point for the energy loss of interest. However, such points are not strongly illuminated by the focused beam arising from the pre-specimen lens system, and therefore are suppressed in strength.

To allow better understanding, an embodiment of the present invention will now be described by way of non-limitative example with reference to the accompanying drawings, in which: Fig.l is a schematic diagram of a scanning confocal electron microscope and associated control apparatus;

Fig. 2 is a flow chart of a method of control implemented in the control apparatus;

Figs. 3a to 3c are schematic diagrams of the confocal scanning electron microscope in different modes of operation; Fig. 4 is an EELS spectrum of a carbon K-edge;

Figs. 5a to 5c are experimental images acquired using a scanning confocal electron microscope;

Figs. 6 to 8 are graphs of the theoretical PSF and Z-response of a scanning confocal electron microscope; Fig. 9 is a graph of FWHM of the Z-response of a scanning confocal electron microscope against the width of an energy selecting slit of an energy filter arranged therein; and

Fig. 10 is a graph of optical sectioning results of inelastic SCEM and inelastic STEM of a flat carbon film against the width of an energy selecting slit of an energy filter arranged in the microscope.

A confocal scanning electron microscope 1 and a control apparatus 2 for controlling the microscope 1 to provide inelastic SCEM are shown in Fig. 1.

The microscope 1 has a conventional construction comprising the following components aligned along an optical axis O. The microscope 1 comprises an electron source 3 which is operable to emit electrons with a variable incidence energy, typically but without limitation of the order of lOOkeV to lOOOkeV. To provide energy resolution, the emitted electrons have a narrow bandwidth of energies as compared to the features of the energy spectrum of the scattering interactions with a sample, such that they may be considered as being of a single energy.

The microscope 1 comprises a pre-specimen lens system 4 operable to focus electrons emitted by the electron source to a point 5. Of course, notwithstanding the description as a "point", the transfer function of the pre-specimen lens system 4 causes the point 5 has some finite spread, the size of which may be characterised by a FWHM. The pre-specimen lens system 4 comprises at least one lens 6 and a spherical aberration (SA) corrector 7.

A specimen 8 is mounted on an actuator 9 that controls the position of the specimen 8. The actuator 9 may take any suitable form for positioning the specimen to sufficient accuracy to match the imaging resolution of the microscope 1, for example being a piezoelectric actuator.

The microscope 1 comprises a detector 10 having an aperture 11 in a physical mask at the image plane. The detector 10 in operation detects all electrons that are incident at the localised region of the aperture 11. The detector 10 may then be a single pixel detector.

Alternatively, the aperture 11 may be artificially constructed using a detector 10 positioned at the image plane, having a wide electron-sensitive area. In this case the signal output from a localised region that is a partial area of the electron-sensitive area is used, this being the signal representative of electrons incident at that localised region.

The detector 10 may be of any suitable construction. The detector 10 typically has a wide detection energy bandwidth capable of scattered electrons of any energy for inelastic SCEM.

The microscope 1 comprises a post-specimen lens system 12 also operable to focus electrons, hi a confocal configuration, the post-specimen lens system 12 focuses electrons from the point 5 onto the aperture 11 of the detector 10. Again, the transfer function of the post-specimen lens system 12 has a spread that may be characterised by a FWHM. The post-specimen lens system 12 comprises at least one lens 13 and an SA corrector 14. hi operation, the lenses 6 and 13 of the pre-specimen and post-specimen lens systems 4 and 12 have the primary effect of focussing the electrons and the SA correctors 7 and 14 of the pre- specimen and post-specimen lens systems 4 and 12 have the primary effect of correcting the spherical aberration of the lenses 6 and 13. The lenses 6 and 13 and SA correctors 7 and 14 have conventional constructions, consisting in essence of electromagnets. They require complex but routine tuning to achieve a confocal configuration in which the focus of the pre-specimen and post-specimen lens systems 4 and 12 are aligned, for example as described in Nellist et al., Applied Physics Letters 89, 124105 (2006).

It is noted that the microscope 1 does not include an energy filter, but is still able to perform inelastic SCEM as described below. It is also noted that no correctors or other measures are included to correct chromatic aberration with the result that both of the pre-specimen and post-specimen lens systems 4 and 12 do have chromatic aberration.

The control apparatus 2 is arranged as follows. The control apparatus 2 comprises a source module 15 arranged to control the electron source

3 to vary the incidence energy of the electrons emitted therefrom, in particular by controlling the accelerating voltage of the electron source 3.

The control apparatus 2 comprises a positioning module 16 arranged to control the actuator 9 to control the position of the specimen 8. The positioning module 16 may control the actuator 9 to scan the specimen 8 in lateral directions perpendicular to the optical axis O to obtain a 2D image of a section of the specimen. The positioning module 16 may control the actuator 9 to scan the specimen 8 in the depth direction along the optical axis O to obtain a 2D image of a section of the specimen.

Alternatively, scanning in lateral and/or depth directions could be achieved by control of the pre- specimen and post-specimen lens systems 4 and 12, as is known in the art. The control apparatus 2 comprises a pre-specimen lens module 17 and a post-specimen lens module 18 arranged to control the pre-specimen and post-specimen lens systems 4 and 12, respectively. In particular, the pre-specimen and post-specimen lens modules 17 and 18 control the power supplied to the electromagnets of the pre- specimen and post-specimen lens systems 4 and 12 to tune their deflection of electrons. The control apparatus 2 comprises a controller 19 that is arranged to provide overall control of the source module 15, positioning module 16 and pre-specimen and post-specimen lens modules

17 and 18.

Lastly, the control apparatus 2 comprises an imaging module 20 supplied with the signal output from the detector 10 and arranged to process the signal to generate and store image data as the specimen 8 is scanned, in this example under the control of the positioning module 16. The imaging module 20 may also provide manipulation and display of the image on a display 21.

The various components of the control apparatus 2 may be implemented by one or more processors executing an appropriate program. Typically, the modules 15 to 18 and 20 might be implemented in separate computers such as PCs, although this is not essential and any of the modules 15 to 18 or 20 may be implemented on the same computer. Similarly, the controller 19 may be implemented in the same computer as one of the modules 15 to 18 or 20 or in its own separate computer.

The control apparatus 2 controls the microscope to detect electrons scattered elastically from the specimen with an energy loss of interest, that may be set by user input to the control apparatus 2. This is achieved without an energy filter to select the energy of the electrons detected by the detector

3, and without correcting for chromatic aberration but instead making use of the chromatic aberration of the post-specimen lens system 12 as will now be explained. As a comparative example, Fig. 2a illustrates the microscope 1 in a confocal configuration but with correction of chromatic aberration. In this case, both elastically and inelastically scattered electrons are deflected by the same amount and so are both focussed onto the aperture 11 of the detector 10 as illustrated by the solid line. Electrons scattered from elsewhere are focussed at a different focal depths, as shown by the broken line, and so do not significantly contribute to the detected signal. However, it would be necessary to include an energy filter to selectively detect elastically scattered electrons experiencing a given energy loss of interest.

In contrast, Fig. 2b illustrates the microscope 1 taking into account the chromatic aberration. In this case, elastically scattered electrons are focused onto the aperture 11 of the detector 10 as illustrated by the solid line. However, as the chromatic aberration causes electrons of different energies to be focussed at different focal lengths, inelastically scattered electrons are focussed at a different focal length, as shown by the broken line, and so do not significantly contribute to the detected signal. In order to detect electrons experiencing the energy loss of interest, this is achieved by the control apparatus 2 configuring the microscope 1 so that the post-specimen lens system 12 in respect electrons of the energy loss of interest below the incidence energy of electrons emitted by the electron source 2 is confocal with the pre-specimen lens system 4 in respect of electrons of the incidence energy.

In principle, this could be achieved by separately tuning the pre-specimen lens system 4 and the post-specimen lens system 12, for example by re-tuning the post-specimen lens system 12 from the configuration shown in Fig. 2b to instead focus electrons of the energy loss of interest below the incidence energy onto the aperture 10 of the detector. However, in practice it is difficult to achieve a confocal condition in this manner because of the complexity involved in simultaneously tuning and aligning both the pre-specimen and post-specimen lens systems 4 and 12 to achieve the confocal configuration, due in part to their inherent magnetic hysteresis. Accordingly, the control apparatus 2 implements a control method illustrated in Fig. 3 that achieves the confocal configuration in an advantageous manner.

In a first step Sl, the electron source 3 is controlled to emit electrons with a first incidence energy. Then, the confocal electron microscope is configured so that the post-specimen lens system 12 in respect electrons of the first incidence energy is confocal with the pre-specimen lens system 4 also in respect of electrons of the first incidence energy. This is done by controlling the pre-specimen and post-specimen lens modules 17 and 18 to simultaneously tune the pre-specimen and post- specimen lens systems 4 and 12 about the mutual optical axis O. This may be achieved using conventional techniques, for example as described in Nellist et al., Applied Physics Letters 89, 124105 (2006). This has the result that the pre-specimen and post-specimen lens systems 4 and 12 are confocal at the point 5 which is at position a in Fig. 2b. In order to illustrate this, Fig. 4 shows the EELS (electron energy loss spectroscopy) spectrum of a carbon K-edge in this configuration illustrating that zero loss in the spectrum is sitting at the confocal point z=0. An experimental image of the probe image in vacuum is formed in this configuration is shown in Fig. 5a.

In a second step S2, the electron source 3 is controlled to emit electrons with a second incidence energy greater than the first incidence energy by an energy offset. This energy offset is selected so that the post-specimen lens system 12 in respect of electrons of the energy loss of interest below the second incidence energy is confocal with the pre-specimen lens system 4 in respect of electrons of the second incidence energy. In particular, the energy offset is equal to the energy loss of interest AE_loss multiplied by a factor &=C2/(C7+C2), where Cl is the chromatic aberration coefficient of the pre-specimen lens system and C2 is the chromatic aberration coefficient of the post- specimen lens system. For example if pre-specimen and post-specimen lens systems 4 and 12 have the same chromatic aberration, for example if they have a substantially identical construction, then the factor is Vi. This factor k results in the confocal configuration being achieved for elastic scattered electrons experiencing the energy loss of interest.

In particular, the pre-specimen lens system 4, due to its chromatic aberration, now focuses the electrons of the second incidence energy emitted from the electrons source 3 onto the point 5 at position b shown in Fig. 2c. At the same time, the energy loss in the specimen 8 causes the post- specimen lens system 12, due to its chromatic aberration, to focus the electrons of the energy loss of interest ΔE_tej below the second incidence energy from that point 5 at position b onto the aperture 11 of the detector 10, again as shown in Fig. 2c. During step S2, the pre-specimen and post-specimen lens systems 4 and 12 are not adjusted to change the degree of deflection so that the focal length in respect of electrons of a given energy remains unchanged. On the basis of an assumption that the spherical aberration, or other parasitic aberrations, introduced by the pre-specimen and post-specimen lens systems 4 and 12 does not change over the change of energy from the first to second incidence energies, then this step S2 may be performed without adjusting the pre-specimen and post-specimen lens systems 4 and 12 at all. To achieve better accuracy, the step S2 may additionally comprise adjusting or tuning the SA correctors 7 and 14 and/or the lenses 6 and 13 of the pre-specimen and post-specimen lens systems 4 and 12 to compensate for the change in spherical aberration or other parasitic aberrations.

In this manner, the confocal configuration achieved in step Sl is maintained in step S2 without adjusting either one of the pre-specimen and post-specimen lens systems 4 and 12, and their chromatic aberration is used to obtain the desired confocal configuration for elastic scattering of electrons with the energy loss of interest ΔE_loss simply by adjustment of the energy source 3. To illustrate this Fig. 4 shows how the resultant ΕΕLS spectrum for a carbon K-edge in the case that the factor k=2 has a blue-shift by Δε = k.AE_{c κ}__edge in the energy regime and how the confocal point

Cc shifts by an amount Δz = —k.AE_{c κ}__edge . — in the position regime.

^Eo As the point 5 on which the electrons are focused has now shifted to position b, in step S3, the actuator 9 is controlled to position the specimen 8 with the desired point of the specimen 8 to be imaged at that position b. Step S3 is performed without adjusting the pre-specimen and post- specimen lens systems 4 and 12 so as to maintain the confocal configuration achieved in step S2. Li this position of the specimen 8, the detector 10 is used to detect the inelastically scattered electrons experiencing the energy loss of interest ΔE_loss , the output signal of the detector 10 being supplied to the imaging module 20.

Subsequently as part of step S3, the actuator 9 may be controlled to position scan the specimen 8 in order to position a scanning sequence of desired points of the specimen 8 to be imaged at that position b. In each position of the specimen 8, the detector 10 is used to detect the inelastically scattered electrons experiencing the energy loss of interest AE_loss , the output signal of the detector 10 being supplied to the imaging module 20 to build up an image.

This method of detecting inelastically scattered electrons has been described as being implemented in the controller 19. As alternative, the method could instead be implemented by the user manually controlling the various modules 15 to 18 and 20.

Thus, the control method allows the performance of inelastic SCEM without the need for an energy filter and without the need to correct the chromatic aberration of the pre-specimen and post- specimen lens systems 4 and 12, by instead using the chromatic aberration of the post-specimen lens system 12 to provide selective detection of inelastically scattered electrons experiencing the energy loss of interest AE_loss .

A related point is that the energy resolution of the detection of inelastically scattered electrons by the detector 10 is dependent on the degree of chromatic aberration of the post-specimen lens system 12. As a result, optionally it is possible to implement control of the energy resolution by varying the chromatic aberration of the post-specimen lens system 12. This may be achieved by the post-specimen lens system 12 further comprising a chromatic aberration controller 22 that is controlled by the post-specimen lens module 18 so that the controller 19 can also control the degree of chromatic aberration.

The above methodology assumes that defocus varies solely as a function of energy loss, and that other aberrations are unchanged. In some practical apparatus, this may be an approximation that reduces the achievable depth and energy resolution, but the methodology is still valid subject to that approximation.

The efficacy of the chromatic aberration in providing energy selectivity is demonstrated by experiments and theoretical calculations as follows.

Experiments have been performed using the Oxford- JEOL 2200FS instrument that is a 200keV confocal scanning electron microscope of the same type as the microscope 1 of Fig. 1. However, the experiments were performed additionally providing an in-column Ω energy filter between the specimen 8 and the detector 9. The microscope 1 was set up using the method of Fig. 3 to study a specimen 8 consisting of a flat amorphous carbon film with a thickness of 20-30nm. The energy filter was arranged to filter the electrons to selectively pass electrons of a desired energy controlled by the position of a slit, the bandwidth of the passed electrons being controlled by the width of the slit. The slit was centred with the size of the slit AE_sι_U at the energy loss of interest ΔE_{bss •} ^so ^^{at on}ty electrons with that energy loss pass through the filter. In this configuration, the microscope formed a focused inelastic probe image at the image plane, the probe image being formed with an 5eV energy-selecting slit placed at carbon K-edge (287.5-292.5eV). The resultant inelastic probe image recorded at the carbon K-edge is shown in Fig. 5b and shows the same diameter and symmetry as the image formed with vacuum shown in Fig. 5a. By way of comparison, Fig. 5c shows the inelastic probe image recorded at the carbon K-edge where the specimen 8 is 56nm over the confocal point 5.

The Z-response of such inelastic SCEM was compared with inelastic STEM. An inelastic SCEM confocal mode for the energy loss of 29OeV by increasing the incidence energy E₀ by an energy offset of 145eV (the factor k for the microscope 1 being Vi in order to maximize the inelastic SCΕM signal from the carbon K-edge. The specimen 8 was moved along the z-axis using the actuator 9 with a step size of 9.3nm. A series of the probe images were recorded on a detector 10 being a 4kx4k Gatan CCD camera for each step. An artificial collector aperture 11 of diameter 0.4nm was applied to the probe images by integrating the intensities within the aperture centred at each probe image. Fig. 10 plots ID sectionings of the specimen 8 for different size of the slit illustrated by the continuous lines, whose the intensities are normalised to have the same maximum. A sectioning using ΕΕLS in the STEM mode (that is using the microscope 1 without the post-specimen lens system 12 was acquired on the same specimen 8 with a 2OeV energy-selecting slit as shown by the broken line in Fig. 10. As predicted by the theoretical Z-Response calculations below, the STEM does not give any thickness determination for this specimen 8 which is an example of an extended object. By adding the post-specimen lens system 12 to operate in inelastic SCEM mode, depth information about the laterally extended carbon film is shown to be determined. With increasing the size of the slit from IeV to 4OeV, the FWHM of the optical sectioning can be seen to be relatively insensitive to the energy filtering window. The focal spread of the post-specimen lens system 12 due to chromatic aberration would be approximately 300 nm, yet the observed depth resolution is better than this. Thus, it is demonstrated that the experiment is not sensitive to the size of the energy window of the energy filter. A corollary of this is therefore that no energy filter is actually required to do the experiment, i.e. that inelastic SCEM can be performed without an energy filter.

A theoretical justification of the efficacy of the chromatic aberration in providing energy selectivity has been derived as follows. In particular, the theoretical PSF and Z-response of the SCEM is derived. To quantitatively examine the effective depth and energy resolutions of inelastic SCEM. For a completely incoherent scattering object function V(R) and in the completely confocal situation, we can write the SCEM image intensity as

PSF(R_x )

=

where PSF is the confocal point spread function (PSF) which describes how the detected image of a point-like scattering object. PSF_x and PSF₂ are the PSFs of the upper and lower column optics, respectively. Fig. 6 shows the PSF with a FWHM of about 5.52nm for the Oxford- JEOL 2200FS instrument, with 22 mrad apertures in the both upper and lower columns. The 'plane spread function' or Z-response describes how a lateral plane is blurred. For an infinitely thin incoherent scattering plane, V (R) is not dependent on the position R and can be take out the integral in Eq. 2.

Taking R_x = 0 , Eq. 2 can be simplified as

The Z-response for different depths z is calculated as Z_SCEM(z) =

(5)

Fig. 6 shows that the theoretical Z-response of the instrument has a FWHM of about 5.9 nm. Eq. 5 can be reduced to the Z-response of STEM as

Z_STEM(z) =

(6)

Because the integrated probe intensities propagating at each plane perpendicular to z direction are constant, the Z-response for STEM is flat line, which indicates the STEM does not provide the depth determination for an extended object.

In the inelastic SCEM mode, the accelerating voltage E₀ is increased by a factor k of the

Cc energy loss k.ΔE, and the confocal point is shifted downwards by Δz = kΛE, . — as shown in

the Fig. 4, so the PSF of inelastic SCΕM is calculated as PSF(R_x ,Az) = \P_x(R -R_x ,z -Az)\²\P₂(R_x -R -(z -Az))\² (7)

It is worth pointing out that the probe function P_x is for the pre-specimen electrons + k.AE_loss , while P₂ is for the post-specimen electrons of energy E_HT — k.AE_loss .

In order to take the chromatic focus spread into account in the PSF due to the residual chromatic aberration in the lower column, we use an energy selecting slit with a size of ΔE_jΛV in the lower column, which can be expressed by a top hat function Ε as

in the energy regime ε , or

in the position regime z. The FSFPSF₂ of the lower column with the chromatic focus spread is a convolution of

E(z) and PSF₂ . Therefore, the total PSF with an energy selecting slit AE_slu is given by

PSF(R₁₅Az)

-ZJ₁₅Z -Az)I²IP₂(R₁ - R₅-(Z -Δz))f ®E(z -Az) (10) where E(z - Az) means that a top hat function is centred at the new confocal point Δz after the E_m is increased by k.AE_loss as shown in the Fig. 4. Meanwhile, we can translate the longitudinal coordinate of the PSF from the position regime

Cc

(z) to the energy regime ( ε ) via the relationship of z = — ε .

x|P₂(R, -R,-(ε-k.AE_lojf ®E(ε-k.AE_loJ(U)

Let S(ε ) be the spectrum function. The effective inelastic SCΕM (taking into account an energy spectrum) confocused at a desire energy loss AE_loss selected by a ΔE_dil energy slit is

in the energy regime and equivalently can be written as

PSF(R, , Az) = |/^» (R - R_x , z - Azf x {\P₂ (R₁ - R,-(z - Az)f <g> [E(z - Δz) x S(z + Δz)]} (13) in the position regime, where S(z + Δz) means that the entire spectrum is shifted upwards by Δz with respect to the original confocal point. This allows the signal at energy loss AE_loss to be selected by the new confocal point as shown in Figs. 2 and 4. The Z-response is given by

Z_SCEM (R, ,Az) = - R,,z - Az)f x {|P₂ (R₁ - R -(z - Az)f ® [E(z -Az) x S(z + Δz)]}dR

= $ PSF₁ x PSF₂ ^'dR

Figs. 7 and 8 show the energy-selected spectrum function S , the PSF and the Z-response with a 14eV energy-selecting slit E with the confocal point at the zero loss and carbon K-edge, respectively, plotted in both the energy and position regimes.

In the position regime, the FWHMs of the PSF and Z-response are about 6.3 & 7.3 nm, and 7.7 & 9.6 nm for the zero loss and the carbon K-edge, respectively in the position regime. It is surprising that with such large energy-selecting slit the SCΕM does not lose its depth resolution and especially the Z-response for the extended object. In order to examine the effect of the energy- selecting slit E , we take out spectrum function and calculated the FWHM of the Z-response as a function of E as shown in the Fig. 9. With increasing the size of the slit, the FWHM increases from 5.9 nm to about 10 nm and flattens out. This is a notable result that the Z-response of SCEM is not deteriorated too much by such large chromatic focal spread in the lower column and loses the depth determination entirely. An extreme case of not using an energy-selecting slit makes the lower column

PSF₂ lose the longitudinal resolution entirely, however the transverse resolution is still preserved. With such high transverse resolution, the intensity of the upper PSF₁ is weighted before being integrated at each plane perpendicular to the z-axis, which allows the Z-response to still hold the depth resolution. The depth resolution of about 10 nm in the worst case is still much better than the resolution of the optical microscopy, which is good for most of the application in biomaterial science. It is even more notable in the energy regime that the FWHMs of the PSF and Z-response are narrower than that of the zero loss peak about 1.5eV (energy resolution of the in-column energy filter) at the current Cc as shown in Fig. 7). The energy resolution is proportional to the FWHM of the pre- specimen PSF and inversely to C2 as expressed by

FWHM_energy = ^FWHMP"^>-^™^E° ₍₁₅₎

So the larger C2, the instrument has the better energy resolution it does. It is traded off with the worse longitudinal resolution of the lower column, but the total longitudinal resolution can not be worse than that of the upper column theoretically. In the current instrument with a FWHM _position of 0.59nm and a C2 of 1.4 mm, FWHM_energy can be achieved to 0.84eV, which is about the energy resolution that the current energy filter can achieve.

Fig. 8 shows that the PSF and Z-response strongly peak at the desired energy loss of 290 eV so than only the electrons within the energy resolution of the PSF and Z-response will be selected at the confocal point. This endues the SCEM with a natural energy-selecting capability, without the external help from the energy filter component.

Therefore, the chromatic aberration does not limit the depth determination of the SCEM for an extended object. In fact, the chromatic aberration is an advantage to select an energy loss of interest and determines the energy resolution, so it acts as an energy filter and makes inelastic SCEM work without an energy filter component in the lower column. With the current energy resolution, it is potentially capable of selecting subtle features in a spectrum to reveal chemical information, bonding information and so on of materials in the 3D SCEM imaging.

Because the energy resolution of the natural selection of the SCEM is comparable or smaller than the energy-selecting slit E we used, E is negligible in the Eq. 12. The PSF of the SCEM without an energy filter fitted in lower column can thus be written as PSF(R₁AE₁₀J = IP₁(R - R

^ε- x {JP₂(Λ, - R ,-(ε- ®[S(ε + AE_los,)]} (16) It is worth pointing out that without the energy-selecting slit E the zero loss peak in the spectrum function S is allowed to be involved in the calculation and its intensity is much stronger than the rest of the spectrum. It acts as additional ID Gaussian function at — k.AE_loss in the energy regime competing with the PSF at k. AE₁₀₁₁ . However, the tails of the PSF quickly decrease to zero at the distance of 1.5 eV from the central peak, so the zero loss peak will be weighted out for a point scatter. But for Z-response, AE_loss can not be chosen too close to the zero loss peak.

For an instrument fitted with a chromatic aberration corrector in the lower column, where Cl is zero and Az = 0 , the position regime for the energy slit function E and spectrum function S no longer exists and therefore the entire spectrum collapses into the elastic confocal point z = 0 , then the PSF in the Eq. 13 is reduced to Eq. (3), which is the normal elastic SCEM PSF . In such condition, ironically in-column energy filter has to be used to select a desired energy loss and the energy resolution is then dependent on the energy selecting slit.

Claims

1. A method of operating a confocal electron microscope to detect electrons scattered inelastically from a specimen with an energy loss of interest, the confocal electron microscope comprising: an electron source for emitting electrons with an incidence energy; a pre-specimen lens system for focussing the electrons emitted from the electron source to a point in a specimen; an electron detector for detecting electrons incident at a localised region; and a post-specimen lens system for focussing electrons scattered from the point onto the localised region, wherein the post-specimen lens system has chromatic aberration, the method comprising configuring the confocal electron microscope so that the post- specimen lens system in respect of electrons of the energy loss of interest below the incidence energy is confocal with the pre-specimen lens system in respect of electrons of the incidence energy.

2. A method according to claim 1 , wherein the electron source is controllable to vary the incidence energy, and the method of configuring the confocal electron microscope comprises: controlling the electron source to emit electrons with a first incidence energy and configuring the confocal electron microscope so that the post-specimen lens system in respect electrons of the first incidence energy is confocal with the pre-specimen lens system in respect of electrons of the first incidence energy; and controlling the electron source to emit electrons with a second incidence energy greater than the first incidence energy by an energy offset selected so that so that the post-specimen lens system in respect electrons of the energy loss of interest below the second incidence energy is confocal with the pre-specimen lens system in respect of electrons of the second incidence energy.

3. A method according to claim 2, wherein the energy offset is equal to the energy loss of interest multiplied by a factor C2/(C1+C2), where Cl is the chromatic aberration coefficient of the pre-specimen lens system and C2 is the chromatic aberration coefficient of the post-specimen lens system.

4. A method according to claim 2 or 3, further comprising positioning a specimen to position a desired point or a scanning sequence of desired points in the specimen at the point at which the pre- specimen lens system focuses electrons emitted from the electron source with the second incidence energy.

5. A method according to any one of the preceding claims, wherein the post-specimen lens system is controllable to vary its chromatic aberration, and the method further comprises controlling the post-specimen lens system to vary its chromatic aberration to vary the energy resolution of the detection of inelastically scattered electrons by the electron detector.

6. A method according to any one of the preceding claims, wherein the pre-specimen lens system has chromatic aberration.

7. A method according to any one of the preceding claims, wherein the pre-specimen lens system and the post-specimen lens system each include at least one lens and a spherical aberration corrector for correcting spherical aberration of the at least one lens.

8. A method according to any one of the preceding claims, wherein the method is performed without using an energy filter arranged between the specimen and the electron detector

9. A control apparatus for controlling a confocal electron microscope that comprises: an electron source for emitting electrons with an incidence energy; a pre-specimen lens system for focussing the electrons emitted from the electron source to a point in a specimen; an electron detector for detecting electrons incident at a localised region; and a post-specimen lens system for focussing electrons scattered from the point onto the localised region, wherein the post-specimen lens system has chromatic aberration, the control apparatus being arranged to control the confocal electron microscope to detect electrons scattered inelastically from a specimen with a predetermined energy loss by configuring the confocal electron microscope so that the post-specimen lens system in respect electrons of the predetermined energy loss below the incidence energy is confocal with the pre-specimen lens system in respect of electrons of the incidence energy.

10. A control apparatus according to claim 9, wherein the control apparatus is capable of controlling the electron source to vary the incidence energy, and the control apparatus is arranged to control the confocal electron microscope to detect electrons scattered inelastically from a specimen with a predetermined energy loss by: controlling the electron source to emit electrons with a first incidence energy and configuring the confocal electron microscope so that the post-specimen lens system in respect electrons of the first incidence energy is confocal with the pre-specimen lens system in respect of electrons of the first incidence energy; and controlling the electron source to emit electrons with a second incidence energy greater than the first incidence energy by an energy offset selected so that so that the post-specimen lens system in respect electrons of the predetermined energy loss below the second incidence energy is confocal with the pre-specimen lens system in respect of electrons of the second incidence energy.

11. A control apparatus according to claim 10, wherein the energy offset is equal to the predetermined energy loss multiplied by a factor C2/(C1+C2), where Cl is the chromatic aberration coefficient of the pre-specimen lens system and C2 is the chromatic aberration coefficient of the post- specimen lens system.

12. A control apparatus according to claim 10 or 11, wherein the control apparatus is capable of controlling the position of a specimen in the confocal electron microscope and is arranged to position the specimen to position a desired point or a scanning sequence of desired points in the specimen at the point at which the pre-specimen lens system focuses electrons emitted from the electron source with the second incidence energy.

13. A control apparatus according to any one of claims 9 to 12, wherein the post-specimen lens system is controllable to vary its chromatic aberration, and the method further comprises controlling the post-specimen lens system to vary its chromatic aberration to vary the energy resolution of the detection of inelastically scattered electrons by the electron detector.

14. A control apparatus according to any one of claims 9 to 13, wherein the pre-specimen lens system has chromatic aberration.

15. A control apparatus according to any one of claims 9 to 14, wherein the pre-specimen lens system and the post-specimen lens system each include at least one lens and a spherical aberration corrector for correcting spherical aberration of the at least one lens.

16. A combination of a control apparatus according to any one of claims 9 to 15 with a confocal electron microscope that the control apparatus is arranged to control.

17. A combination according to claim 16, wherein the confocal electron microscope does not include an energy filter arranged between the specimen and the electron detector.