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Contrast transfer function (CTF) correction (zz)  

2013-09-05 22:25:53|  分类: EM |  标签: |举报 |字号 订阅

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Contrast transfer function (CTF) correction
 
What is the contrast transfer function?

The image formation in bright field electron microscopy can be described by the action of the contrast transfer function (CTF) H(k). Accordingly, the relationship between the object o(r) and the image contrast i(r) can be written as i(r) = o(r)* h(r), where * stands for the convolution operation, and h(r) is the point spread function, which is the Fourier transform of H(k). Thus, following the convolution theorem, I(k) = O(k)H(k).

The shape of the CTF, H(k), depends on several parameters (for details, see Frank, 2006):

defocus [A] - which describes the deviation in the focus of the objective lens from the "Gaussian focus."
spherical aberration coefficient [mm] - which describes the (third order) spherical aberration of the wave front in the objective lens.
source size [1/A] - which describes the illumination divergence, expressed as a size in the back focal plane (hence a quantity in reciprocal space).
defocus spread - which describes the spread of defocus due to the spread of electron energies or to the fluctuation of lens current.

The only parameter being varied in the experiment is the defocus. Depending on the defocus setting, different features of the object appear enhanced or suppressed in the image. This is because the CTF oscillates between -1 (negative contrast transfer) and +1 (positive contrast transfer) as we go from low to high spatial frequencies. The exact locations of the zero crossings (where no contrast is transferred, and information is lost) depends on the defocus. [For examples of CTFs at different defocus settings, click here.]


CTF correction

In CTF correction, we attempt to retrieve the undistorted object from the image. This attempt is compromised by the presence of noise; i.e., recovery of the object is never ideal. Since the CTF always has zero crossings, part of the information about the object is lost. This is why we make use of several images obtained at different defocus settings, hoping that the resulting CTFs Hn(k) jointly (after appropriate weighting) cover the whole Fourier space without gap.

The Wiener filter is the least square solution to the problem of signal recovery in the presence of noise. Let's assume we have N images in (r) (with Fourier transforms In(k)) whose CTFs are Hn(k). In that case, the best estimation of the object transform O(k) is Contrast transfer function (CTF) correction (zz) - dingding830106 - Wei Ding blogwhere Contrast transfer function (CTF) correction (zz) - dingding830106 - Wei Ding blogand SNR is the signal-to-noise ratio, defined as the ratio of signal to noise variances.


Example of CTF correction

In the SPIDER procedure file, ctfexample.spi, we first simulate the action of the electron microscope, by applying a CTF to an "object", which is the projection of the 3D density map of the ribosome, and adding noise to the result, and then use the 2D Wiener filtering operation described above to retrieve the original. The gallery of images describes the progress of these operations. Note that the choice SNR=100 makes the Wiener filter quite aggressive, and this benefits the recovery of low spatial frequencies responsible for defining the particle's boundary and overall shape.

Contrast transfer function (CTF) correction (zz) - dingding830106 - Wei Ding blog


CTF parameters in SPIDER and single particle reconstruction

Various CTF parameters are used in SPIDER's TF operations. These parameters, along with some others, are listed in a parameter document file. (See also the glossary for definitions.)

In single particle reconstruction,
several procedure files: 1) compute the power spectra of the micrographs, 2) estimate defocus, and 3) assign micrographs to defocus groups.
CTF parameters are estimated with the the SPIDER operation TF ED.
CTF correction is applied during 3D reconstruction, via the TF CTS operation.
In this approach, 3-dimensional CTF correction is carried out for each defocus group, creating a volume for each defocus group. These volumes are combined to form a single, CTF-corrected volume. For details, see Penczek et al.,1997.


CTF graphical tools

A number of graphical tools are included with the SPIRE distribution:
ctfdemo.py : a graphical interface that lets you experiment with the various CTF parameters used in SPIDER.
ctfmatch.py : a tool for analyzing the output from SPIDER's TF ED operation.
ctfgroup.py : graphically assign micrographs to defocus groups.



References

Frank, J. (2006) Three-Dimensional Electron Microscopy of Macromolecular Assemblies. Oxford University Press, New York.

P.A. Penczek, J. Zhu, R. Schr鰀er, J. Frank (1997) Three Dimensional Reconstruction with Contrast Transfer Compensation from Defocus Series Special Issue on Signal and Image Processing, Scanning Microscopy Volume 11, 1997, page 147.
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