pixels, i.e., doubling the Nyquist frequency [38]. This means that if the electron flux
is kept slow relative to image acquisition rate, detections events can be interpreted as
having come from a single electron, enabling equal weighting of each electron
collision with the detector, resulting in lowered noise, a strategy referred to as
counting mode. The very high DQEs of new DDDs enables acquisition of images
with superior quality to CCD camera images with considerably lower electron dose
resulting in less electron damage to the specimen. The DQEs of current DDDs are
superior to both film and CCDs at all spatial frequencies [39]. Each camera has
different imaging properties which may be optimal for different specimens and
techniques. If DDDs continue to improve on frame-rate and precision of the
detection of individual electron events, their DQEs are expected to approach 100%,
even at spatial frequencies approaching Nyquist. The problem then becomes storage
of the large volumes of data that is generated!
Because inelastically scattered electrons are incoherent due to reduced energy,
they contribute only noise to the image, and some contemporary microscopes are
equipped with an energy filter that can remove them—a crucial advantage for
thicker tomography specimens. Energy filterered TEM (EFTEM) allows the
selection of only electrons within a specific range of energy-loss to hit the detector,
greatly increasing the signal-to-noise ratio of images of thicker specimens. Energy
filters are composed of magnetic prisms that bend the electron beam, altering
electron trajectories as a function of their energy loss, enabling filtering of all but a
narrow window of energies by inserting a physical ‘slit’ in the beam path; slit size
and centre can be altered according to user needs.
An alternative approach to generating phase contrast is to use the still relatively
new technology of phase plates which function by altering the phase difference
between the scattered and unscattered electron beam [40]. In recent years, two types
of phase plates have become commercially available. The Zernike phase plate [41]
provides a uniform spectral transfer with a flat CTF for frequencies important for
tomography. The Zernike phase plate is made of a metal aperture supporting a thin
amorphous carbon film with a small hole (*1um) in the centre positioned at the
back focal plane of the objective lens that unscattered electrons pass through.
Scattered electrons are further scattered by the carbon film increasing their path
length, hence producing a phase shift. At the desired phase shift of 90° the sine
component of the CTF shifts to cosine, which maximises contrast for low spatial
frequencies. The more recently developed Volta Phase plate has demonstrated
useful improvements in contrast for ECT [42]. This phase plate is a heated continuous film of amorphous carbon that builds a potential when irradiated by the
focused electron beam. This potential confers a phase shift on the unscattered beam,
resulting in phase contrast. In addition to improved contrast, use of a phase plate
allows for at- or near-focus imaging and reduction in electron dose [43], not only
decreasing specimen damage but reducing specimen movement and allowing for
improved tilt series alignments and improved reconstructions.
70
J. L. Ferreira et al.
is kept slow relative to image acquisition rate, detections events can be interpreted as
having come from a single electron, enabling equal weighting of each electron
collision with the detector, resulting in lowered noise, a strategy referred to as
counting mode. The very high DQEs of new DDDs enables acquisition of images
with superior quality to CCD camera images with considerably lower electron dose
resulting in less electron damage to the specimen. The DQEs of current DDDs are
superior to both film and CCDs at all spatial frequencies [39]. Each camera has
different imaging properties which may be optimal for different specimens and
techniques. If DDDs continue to improve on frame-rate and precision of the
detection of individual electron events, their DQEs are expected to approach 100%,
even at spatial frequencies approaching Nyquist. The problem then becomes storage
of the large volumes of data that is generated!
Because inelastically scattered electrons are incoherent due to reduced energy,
they contribute only noise to the image, and some contemporary microscopes are
equipped with an energy filter that can remove them—a crucial advantage for
thicker tomography specimens. Energy filterered TEM (EFTEM) allows the
selection of only electrons within a specific range of energy-loss to hit the detector,
greatly increasing the signal-to-noise ratio of images of thicker specimens. Energy
filters are composed of magnetic prisms that bend the electron beam, altering
electron trajectories as a function of their energy loss, enabling filtering of all but a
narrow window of energies by inserting a physical ‘slit’ in the beam path; slit size
and centre can be altered according to user needs.
An alternative approach to generating phase contrast is to use the still relatively
new technology of phase plates which function by altering the phase difference
between the scattered and unscattered electron beam [40]. In recent years, two types
of phase plates have become commercially available. The Zernike phase plate [41]
provides a uniform spectral transfer with a flat CTF for frequencies important for
tomography. The Zernike phase plate is made of a metal aperture supporting a thin
amorphous carbon film with a small hole (*1um) in the centre positioned at the
back focal plane of the objective lens that unscattered electrons pass through.
Scattered electrons are further scattered by the carbon film increasing their path
length, hence producing a phase shift. At the desired phase shift of 90° the sine
component of the CTF shifts to cosine, which maximises contrast for low spatial
frequencies. The more recently developed Volta Phase plate has demonstrated
useful improvements in contrast for ECT [42]. This phase plate is a heated continuous film of amorphous carbon that builds a potential when irradiated by the
focused electron beam. This potential confers a phase shift on the unscattered beam,
resulting in phase contrast. In addition to improved contrast, use of a phase plate
allows for at- or near-focus imaging and reduction in electron dose [43], not only
decreasing specimen damage but reducing specimen movement and allowing for
improved tilt series alignments and improved reconstructions.
70
J. L. Ferreira et al.