To understand and interpret a cryo-tomogram it is also important to understand
digital image processing concepts. Centrally important is to have an intuitive
understanding of Fourier transforms, 3-D Fourier space, and how Fourier concepts
can be used to reconstruct a 3-D volume from 2-D projection images taken from
different angles [8–10] (the central projection theorem and its real space equivalent,
the inverse Radon transform), and describing images in terms of their constituent
spatial frequencies. Of critical importance to interpretation is the fact that
cryo-tomograms are invariably ‘missing’ parts of data in Fourier space due to the
fact that the grid cannot be rotated through a full ± 90° during imaging. This
missing data, often referred to as the ‘missing wedge’ (because the missing data is
wedge-shaped in 3-D Fourier space) results in artefacts in the tomogram, manifesting as greatly reduced resolution along the axis parallel to the electron beam,
which can only be overcome using averaging techniques.
Another important digital imaging concept is the number of pixels required to
capture desired details in an image. At low magnifications the image of a structure
of interest covers only a few pixels, but as magnification increases, the image
expands to cover a larger array of pixels. To capture the details that you are
interested in, therefore, your sampling must be sufficiently fine-grained; the highest
resolution (or spatial frequency, in Fourier terms) that can be sampled by a given
pixel size is described by the Nyquist frequency: two objects cannot be resolved if
their separation is smaller than a single pixel; the size of a pixel must therefore be
no more than half that of the desired highest resolution.
3.3 Specimen Preparation for ECT
Vitrification as a preservation method means a specimen can be inserted into the high
vacuum of the electron microscopy column for imaging without artefact-associated
fixatives [11]. The first cryo-tomographic studies were performed in the mid-1990s
[12–14], building on successes in single particle analysis of vitrified specimens [15]
stemming from work on vitrification of water in 1981 [16].
Specimen choice and experimental design is critical in ECT. Unsurprisingly,
ECT adheres to the GIGO principle—“Garbage In, Garbage Out”—i.e., to obtain
good tomography data, an appropriate specimen must be prepared sufficiently well.
Before vitrification, therefore, the specimen that best answers the biological question must be selected. If imaging intact cells, is a wild-type organism sufficient, or
would another strain or species have cells better suited for tomography, or produce
more copies of a specific structure of interest? Should key genes be deleted,
truncated, or otherwise manipulated to perturb cellular or molecular structure? If
dynamic information is important, can these insights be gained from vitrified
specimens, and if so, how could this be achieved?
The most important factor behind image quality is that the specimen and its
envelope of vitreous ice are thin enough to avoid excessive electron scattering,
meaning it is important to consider strategies to image the thinnest possible
3 Electron Cryo-Tomography
65
digital image processing concepts. Centrally important is to have an intuitive
understanding of Fourier transforms, 3-D Fourier space, and how Fourier concepts
can be used to reconstruct a 3-D volume from 2-D projection images taken from
different angles [8–10] (the central projection theorem and its real space equivalent,
the inverse Radon transform), and describing images in terms of their constituent
spatial frequencies. Of critical importance to interpretation is the fact that
cryo-tomograms are invariably ‘missing’ parts of data in Fourier space due to the
fact that the grid cannot be rotated through a full ± 90° during imaging. This
missing data, often referred to as the ‘missing wedge’ (because the missing data is
wedge-shaped in 3-D Fourier space) results in artefacts in the tomogram, manifesting as greatly reduced resolution along the axis parallel to the electron beam,
which can only be overcome using averaging techniques.
Another important digital imaging concept is the number of pixels required to
capture desired details in an image. At low magnifications the image of a structure
of interest covers only a few pixels, but as magnification increases, the image
expands to cover a larger array of pixels. To capture the details that you are
interested in, therefore, your sampling must be sufficiently fine-grained; the highest
resolution (or spatial frequency, in Fourier terms) that can be sampled by a given
pixel size is described by the Nyquist frequency: two objects cannot be resolved if
their separation is smaller than a single pixel; the size of a pixel must therefore be
no more than half that of the desired highest resolution.
3.3 Specimen Preparation for ECT
Vitrification as a preservation method means a specimen can be inserted into the high
vacuum of the electron microscopy column for imaging without artefact-associated
fixatives [11]. The first cryo-tomographic studies were performed in the mid-1990s
[12–14], building on successes in single particle analysis of vitrified specimens [15]
stemming from work on vitrification of water in 1981 [16].
Specimen choice and experimental design is critical in ECT. Unsurprisingly,
ECT adheres to the GIGO principle—“Garbage In, Garbage Out”—i.e., to obtain
good tomography data, an appropriate specimen must be prepared sufficiently well.
Before vitrification, therefore, the specimen that best answers the biological question must be selected. If imaging intact cells, is a wild-type organism sufficient, or
would another strain or species have cells better suited for tomography, or produce
more copies of a specific structure of interest? Should key genes be deleted,
truncated, or otherwise manipulated to perturb cellular or molecular structure? If
dynamic information is important, can these insights be gained from vitrified
specimens, and if so, how could this be achieved?
The most important factor behind image quality is that the specimen and its
envelope of vitreous ice are thin enough to avoid excessive electron scattering,
meaning it is important to consider strategies to image the thinnest possible
3 Electron Cryo-Tomography
65