TRANSMISSION ELECTRON MICROSCOPE
The present invention relates to a transmission electron
microscope.
When imaging non-conducting specimens by transmission electron
microscopy (TEM), beam-induced positive charge builds up on
the specimen due to the ejection of secondary electrons.
Transmission images of such charged specimens are degraded due
to (1) electrostatic perturbation of the imaging optics and
(2) charge-induced movement and modification of the specimen.
These problems are a major limitation to a wide variety of
imaging experiments in biology and materials science,
including the imaging of frozen-hydrated specimens by
cryomicroscopy.
For specimens that are resistant to radiation damage by the
imaging electron beam, images are often recorded after
sufficient pre-exposure such that the positive charge build-up
on the specimen reaches a steady-state because secondary
electrons cannot escape the positive charge.For specimens
that are not resistant to radiation damage, pre-exposure is
not an option, because relevant structural details of the
specimen must be recorded using the first few electrons that
irradiate the specimen.Thus the charge on the specimen can
change for the duration of the exposure.Image degradation by
charging may be most problematic for cryomicroscopy of
biological specimens precisely under imaging conditions that
are otherwise most advantageous for imaging structural detail,
such as when they are suspended in holes over ice, or at
liquid helium temperature where specimen conductivity is
reduced.
Brink et al., Evaluation of charging on macromolecules in
electron cryomicroscopy, Ultramicroscopy, 72 (1998) 41-52
describes charge build up on non-conducting specimens due to
secondary electron emission. In particular, an experiment is
disclosed in which a small diameter beam is used to charge up
a specimen, and a wide diameter beam is then used to observe
the charged area and eventually discharge it. It is suggested
that some of the secondary electrons which are emitted across
the entire region when the specimen is examined with the wide
beam return to compensate the built up positive charge.
Warrington, A simplechargeneutralizer fortheelectron
microscope, J. Sci. Instrum., 43 (1966) 77-78, proposes a
charge neutralizer consisting of an earthed film of vacuum
deposited carbon and aluminium supported above the specimen
plane of the objective lens.The electron beam passes through
the film before striking the specimen to be examined.Low
energy electrons ejected from the film then discharge the non-
conducting specimen.
Beam-induced positive charge can build up on other non-
conducting bodies, such as electron optical elements, located
on the path of the electron beam, and degrade their
performance.Examples of such bodies are phase plates and
electron biprisms.
US 2002/0011566 discloses an antistatic phase plate for use in
phase-contrast electron microscopy, the phase plate being made
of a thin film of conductive amorphous material.
Frost, Image-planeoff-axiselectron holography: low-
magnificationarrangements, Meas. Sci. Technol., 10 (1999)
333-339, discusses measurements of the deflection angle at an
electron biprism which indicate that the biprism fibre is
positively charged by the imaging electron beam.
The present invention aims to overcome or mitigate problems of
beam-induced positive charge build up.
In a first aspect, the present invention provides a
transmission electron microscope (TEM) having:
a target body position on the electron optical axis of the
microscope,
an electrically conductive body off the axis of the
microscope,
an electron source for producing an axial electron beam
which, in use, impinges upon a target body located at the
target body position, and
a system for simultaneously producing a separate off-axis
electron beam which, in use, impinges on the electrically
conductive body causing secondary electrons to be emitted
therefrom;
wherein the electrically conductive body is located such
that the emitted secondary electrons impinge on the target
body to neutralise positive charge which may build up on the
target body.
As used herein, the term "separate off-axis electron beam"
excludes any off-axis electron beam that may be produced by
scattering or diffraction of the axial electron beam.
By producing, simultaneously with the axial electron beam, a
separate off-axis electron beam that causes charge-
neutralising secondary electrons to be emitted, the TEM can be
used e.g. to image a non-conducting specimen while at the same
time operating to reduce charge build-up on the specimen.In
other words, the separate off-axis electron beam and the off-
axis electrically conductive body may be thought of as a
dedicated system for reducing or eliminating charge build-up.
Advantageously, and in contrast to the observations of Brink
et al. ibid., the TEM operator can image a specimen with a
narrow-diameter beam and still avoid problems of beam-induced
positive charge build up.Further, the inconvenience and
disturbance of a charge neutralizer positioned in the path of
the axial electron beam according to the proposal of
Warrington ibid, can be avoided.
Preferably, the off-axis electrically conductive body is
located adjacent to the target body position.The secondary
electrons emitted from the body will then have a relatively
short distance to travel before impinging on the target body,
which can increase the flux of impinging electrons.
Preferably, the off-axis electron beam is a paraxial electron
beam.A paraxial electron beam can be defined as a beam that
is focusable onto the electron optical axis by the TEM lenses,
but has a minimum divergence angle that is greater than the
maximum divergence angle of the axial electron beam.
Conveniently, the off-axis electron beam can be produced by
the same electron source that produces the axial electron
beam, i.e. the system for simultaneously producing a separate
off-axis electron beam can include the electron source.
Advantageously, using this approach, a conventional TEM can
readily be converted into a TEM according to the present
invention.
For example, the system for producing an off-axis electron
beam may comprise an aperture body positioned between the
electron source and the target body position, the aperture
body having an axial aperture for transmission of the axial
electron beam and further having an off-axis aperture for
production of the off-axis electron beam.Such an aperture
body may simply replace an existing condenser aperture body of
an existing TEM.
The system for producing an off-axis electron beam may produce
a plurality of such beams which, in use, impinge on the
electrically conductive body (or, more preferably, respective
electrically conductive bodies).This makes it possible, for
example, to neutralise build up of positive charge at
respective target bodies at spaced positions on the electron
optical axis position.Thus, the aperture body may have a
plurality of off-axis apertures for production of respective
off-axis electron beams, each off-axis electron beam, in use,
impinging on the off-axis electrically conductive body or a
respective off-axis electrically conductive body.
Typically, the TEM has at least one condenser lens between the
electron source and the target body position, and the aperture
body may be positioned between the condenser lens and the
target body position.Thus the aperture can limit the
illuminating field of the condenser lens.
Indeed, in a further aspect, the present invention provides a
multi-aperture aperture body as discussed above.
In other embodiments of the first aspect, the system for
producing an off-axis electron beam may comprise a further
electron source (or a plurality of further electron sources if
a plurality of off-axis electron beams are to be deployed).
This can increase the complexity and cost of the TEM, and may
make it more difficult to convert a conventional TEM into a
TEM according to the present invention.However, a further
electron source for producing the off-axis electron beam can
provide an advantage by allowing the off axis beam intensity
to be varied independently of the axial beam, or for the off-
axis beam to remain constant if the axial beam is pulsed.
Typically, the target body position is a specimen position,
the axial electron beam, in use, impinging upon a specimen,
and the emitted secondary electrons impinging on the specimen
to neutralise positive charge which may build up on the
specimen.Conveniently, the off-axis electrically conductive
body can then be provided by a specimen support which holds
the specimen at the specimen position.
However, the target body, or one of the target bodies, can be
an electron optical element such as a phase plate or an
electron biprism.In such cases the axial beam will typically
impinge on a specimen, and that specimen may be another target
body.When there are plural target bodies, each may have a
respective electrically conductive body.
Embodiments of the invention will now be described by way of
example with reference to the accompanying drawings in which:
Figure 1 shows schematically a non-conducting specimen, and a
TEM axial electron beam used to image the specimen;
Figure 2 shows schematically a longitudinal section through a
TEM according to an embodiment of the invention;
Figure 3 shows schematically a multi-hole aperture;
Figure 4 is a recording on film of axial and paraxial electron
beams;
Figure 5(a) is an image of a seven-hole condenser aperture,
and Figure 5(b) is an electron microscope recording on a CCD
camera of electron beams produced by the seven-hole aperture
designed;
Figure 6 is a recording on a CCD camera of a typical beam-
sensitive (frozen-hydrated) specimen with the positions of
beams indicated for imaging of a specimen using the seven-hole
aperture; and
Figure 7 shows two images of the same area of a typical single
particle specimen in vitreous ice over a hole in a carbon
support (a) with the seven-hole aperture used as a charge
compensator and (b) without the compensator.
Figure 1 shows schematically a non-conducting specimen, and a
TEM axial electron beam (block arrows, e) used to image the
specimen.The electron beam causes secondary electrons (line
arrows, SE) to be ejected from the specimen, leaving the
specimen with a positive charge.This positive charge can
cause electrostatic perturbation of the imaging optics and
also can cause movement and Coulombic explosion of the
specimen.
Figure 2 shows schematically a longitudinal section through a
TEM according to an embodiment of the invention.An axial
electron beam 1 produced by an electron source 2 impinges on a
non-conducting specimen 3 held by specimen holder 4.A multi-
hole aperture 5 positioned between TEM condenser lens 6 and
the specimen has a central hole 7 for the axial electron beam
and an off-centre hole 8 which produces a paraxial electron
beam 9.
The paraxial electron beam irradiates a grounded conductor 10
which is adjacent the specimen but off the axis of the TEM,
the grounded conductor being integral with the specimen
holder.The irradiation of the paraxial electron beam causes
secondary electrons SE to be emitted by the grounded
conductor, and some of these electrons in turn impinge on the
non-conducting specimen to neutralise positive charge which
has built up on the specimen.Thus the paraxial beam and the
grounded conductor act as charge compensator for the specimen.
A similar arrangement (not illustrated) can be used to
neutralise positive charge on other bodies, such as a phase
plate or an electron biprism, which are susceptible to charge
build up due to secondary electron emission.
Figure 3 shows schematically a multi-hole aperture as viewed
along the optical axis of a TEM.The aperture has a central
hole 7 and six off-centre holes 8 circumferentially spaced
around the central hole.Each off-centre hole can produce a
respective paraxial beam.A typical diameter for the holes
could be about 50 µm and typical centre-to-centre spacing for
the holes could be about 200 µm.Such an aperture could be
retrofitted to an existing microscope to allow it to produce
several off-axis paraxial beams.
Figure 4 is a recording on film of the axial and paraxial
electron beams produced using a condenser aperture having a 50
µm diameter central hole and one 100 µm diameter off-centre
hole.
Figure 5(a) is an image recorded on a CCD camera of a seven-
hole condenser aperture according to an embodiment of the
present invention.The image was produced by placing the
seven-hole aperture in the specimen holder of the electron
microscope.Each hole is 50 µm diameter and the offset is 200
µm centre-to-centre.
Figure 5(b) is an electron microscope recording on a CCD
camera of electron beams produced by the seven-hole aperture.
The aperture is arranged to produce axial and paraxial beams
on the specimen.The condenser lens demagnifies the image of
the apertures onto the specimen plane. The paraxial beams are
slightly elliptical compared to the axial beam due to
spherical aberration of the condenser lens system.
Figure 6 is a recording on a CCD camera of a typical beam-
sensitive (frozen-hydrated) specimen with the positions of
beams indicated for imaging of a specimen using the seven-hole
aperture.If a single hole aperture is required in place of
the seven-hole aperture, both apertures can mounted on the
microscope aperture holder so that one or the other can be
shifted into position as needed.Alternatively, an extra
occluding aperture could be inserted to obstruct the paraxial
beams.
The imaging protocol consisted ofthe identification of a
region of interest in the specimen at low magnification (e.g.
10K times) using single beam illumination, focus
determination by imaging the support adjacent to the specimen
at a high magnification (e.g. 200K times) using single beam
illumination, and then image recording of the region of
interest at intermediate magnification (e.g. 60K times with
focus parameters established during focus mode) using either
the single or the seven-hole aperture. In this way the pre-
exposure of the region of interest is minimized.The specimen
is a holey carbon film covered with a thin film of vitreous
ice containing material of interest at liquid nitrogen
temperature (-195°C). The region of interest is material
located in the ice film over any of the holes.As Figure 6
shows, in this case the axial beam position is within a hole.
Figure 6 shows a low magnification image recorded subsequent
to focusing and exposure. The focus position is 2 µm away from
the axial beam position and can be identified as a bright spot
where the concentrated dose has removed a layer of ice from
the carbon.The position of the six off-axis beams on the
surrounding carbon is also indicated. They produce a
characteristic charging "footprint" in the overlying ice
(Brink et al. ibid.). Irradiation of the carbon support at
the paraxial beam positions causes SE electrons to impinge on
the axial beam position located at the thin film of ice over
the hole in the carbon support.
Figure 7 shows two images of the same area of a typical single
particle specimen in vitreous ice over a hole in a carbon
support (a) with the seven-hole aperture used as a charge
compensator and (b) without the compensator. In each case the
axial beam was within the hole and did not irradiate the
adjacent carbon support.In image (a) the paraxial beams from
the compensator irradiated the adjacent carbon support.These
beams were absent in image (b).The reduced image quality in
the absence of the compensator (image (b)) is attributed to
charging of the film resulting in image blurring and
distortion by the mechanisms described above.
While the invention has been described in conjunction with the
exemplary embodiments described above, many equivalent
modifications and variations will be apparent to those skilled
in the art when given this disclosure.Accordingly, the
exemplary embodiments of the invention set forth above are
considered to be illustrative and not limiting.Various
changes to the described embodiments may be made without
departing from the spirit and scope of the invention.
WE_CLAIMS
1. A transmission electron microscope having:
a target body position on the electron optical axis of the
microscope,
an electrically conductive body off the axis of the
microscope,
an electron source for producing an axial electron beam
which, in use, impinges upon a target body located at the
target body position, and
a system for simultaneously producing a separate off-axis
electron beam which, in use, impinges on the electrically
conductive body causing secondary electrons to be emitted
therefrom;
wherein the electrically conductive body is located such
that the emitted secondary electrons impinge on the target
body to neutralise positive charge which may build up on the
target body.
2. A transmission electron microscope according to claim 1,
wherein the off-axis electron beam is a paraxial electron
beam.
3. A transmission electron microscope according to claim 1 or
2, wherein the off-axis electrically conductive body is
located adjacent the target body position.
4. A transmission electron microscope according to any one of
the previous claims, wherein the system for producing an off-
axis electron beam comprises an aperture body positioned
between the electron source and the target body position, the
aperture body having an axial aperture for transmission of the
axial electron beam and further having an off-axis aperture
for production of the off-axis electron beam.
5. A transmission electron microscope according to claim 4,
wherein the aperture body has a plurality of off-axis
apertures for production of respective off-axis electron
beams, each off-axis electron beam, in use, impinging on the
off-axis electrically conductive body or a respective off-axis
electrically conductive body.
6. A transmission electron microscope according to claim 4 or
5, further having at least one condenser lens between the
electron source and the target body position, the aperture
body being positioned between the condenser lens and the
target body position, the aperture body limiting the
illuminating field of the condenser lens.
7. A transmission electron microscope according to claim 1 or
3, wherein the system for producing an off-axis electron beam
comprises a further electron source.
8. A transmission electron microscope according to any one of
the previous claims, wherein:
the target body position is a specimen position,
the axial electron beam, in use, impinges upon a specimen,
and
the emitted secondary electrons impinge on the specimen to
neutralise positive charge which may build up on the specimen.
9. A transmission electron microscope according to claim 8,
wherein the off-axis electrically conductive body is provided
by a specimen support which holds the specimen at the specimen
position.
10. A transmission electron microscope according to any one of
claims 1 to 7, wherein:
the target body position is a phase plate position,
the axial electron beam, in use, impinges upon a phase
plate, and
the emitted secondary electrons impinge on the phase plate
to neutralise positive charge which may build up on the phase
plate.
11. A transmission electron microscope according to any one of
claims 1 to 7, wherein:
the target body position is an electron biprism position,
the axial electron beam, in use, impinges upon an electron
biprism, and
the emitted secondary electrons impinge on the electron
biprism to neutralise positive charge which may build up on
the electron biprism.
12. A transmission electron microscope according to any one of
the previous claims, having a plurality of target body
positions spaced on the electron optical axis of the
microscope, a plurality of respective electrically conductive
bodies off the axis of the microscope, and a system for
simultaneously producing a plurality of separate off-axis
electron beams which, in use, respectively impinge on the
electrically conductive bodies.
13. The aperture body of any one of claims 4 to 6.

A transmission electron microscope has a target body position
on the electron optical axis of the microscope, and an
electrically conductive body off the axis of the microscope.
The microscope also has an electron source for producing an
axial electron beam. In use, the beam impinges upon a target
body located at the target body position. The microscope
further has a system for simultaneously producing a separate
off-axis electron beam. In use, the off-axis electron beam
impinges on the electrically conductive body causing secondary
electrons to be emitted therefrom. The electrically
conductive body is located such that the emitted secondary
electrons impinge on the target body to neutralise positive
charge which may build up on the target body.