CONTRAST TRANSFER FUNCTION (CTF) CORRECTION FOR ELECTRON TOMOGRAPHY

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 对于传统的光学器件（即，在没有相位板的情况下），低剂量冷冻ET中的图像对比度主要通过使显微镜显著散焦（通常散焦3-8微米）来实现。 这种大的散焦将CTF的第一零偏移到低得多的空间频率，并且结果，由于第一零之后的相位反转，所采集的断层图像不具有超过约3-4 nm分辨率的直接可用信息。 CTF校正是在冷冻断层图像中实现2-3 nm分辨率的几种补充方法之一。 我们已经开发了一个GUI程序，可以鲁棒地确定中心散焦（因此CTF的第一个零）的一个子集的意见，从倾斜系列。 我们的方法使用周期图平均计算图像的1D功率谱，然后从中减去显微镜和CCD相机的量子噪声功率谱。 为了提高信噪比，来自不同Z高度处的条带的功率谱被移位成与来自倾斜图像的中心的功率谱近似配准。 用户交互式地设置用于在第一个零点之前和之后拟合曲线的限制，并且这些限制然后可以在通过倾斜角度的范围分析视图的不同子集时应用。 实验表明，计算出的散焦同意与散焦报告的SerialEM程序，在我们的F20和F30显微镜。 第二个程序被写入以在确定中心处的CTF的第一个零点之后反转相位反转。 该校正方法考虑了沿垂直于倾斜轴的方向的散焦梯度沿着。 它将图像细分成平行于倾斜轴的条带，使得条带内的散焦变化与散焦相比很小。 然后，假设从倾斜几何形状计算的恒定散焦，其校正条带。 在这个阶段，我们仍然不能量化的分辨率增益由于CTF校正。我们的计划是首先使用来自一些已知晶体蛋白质的图像来验证我们的校正算法，然后将其应用于我们的断层图像，看看它能在多大程度上改善我们的粒子平均程序的最终结果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. With conventional optics (i.e., in the absence of a phase plate), image contrast in low dose cryo-ET is mainly achieved by defocusing the microscope significantly, typically by 3-8 microns. This large defocus shifts the first zero of the CTF to a much lower spatial frequency, and as a result the acquired tomogram does not have directly usable information beyond about 3-4 nm resolution due to phase inversion after the first zero. CTF correction is one of several complementary approaches to achieve 2-3 nm resolution in cryotomograms. We have developed a GUI program that can robustly determine the center defocus (thus the first zero of CTF) for a subset of views from a tilt series. Our method computes the 1D power spectrum of the images using periodogram averaging and then subtracts from it the quantum noise power spectrum of the microscope and CCD camera. To improve the signal to noise ratio, power spectra from strips at different Z heights are shifted into approximate registration with those from the center of the tilted images. The user interactively sets limits for fitting curves before and after the first zero, and those limits can then be applied when analyzing different subsets of views through the range of tilt angles. Experiments show that the computed defocus agrees well with the defocus reported by the SerialEM program, on both our F20 and F30 microscopes. A second program was written to reverse the phase inversion after the first zero of CTF in the center is determined. The correction method takes into account the defocus gradient along the direction perpendicular to the tilt axis. It tessellates an image into strips parallel to the tilt axis so that defocus variation within a strip is small comparing with the defocus. Then it corrects the strip assuming a constant defocus that is computed from the tilt geometry. At this stage we still can not quantify the resolution gain due to CTF correction. The plan is to first validate our correction algorithm using images from some known crystal protein, then apply it to our tomograms to see how much it can improve the final result of our particle averaging program.