CONTRAST TRANSFER FUNCTION (CTF) CORRECTION FOR ELECTRON TOMOGRAPHY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 用传统的光学系统(即在没有相位板的情况下)，低剂量冷凝器的图像对比度主要是通过使显微镜明显散焦来实现的，通常是3-8微米。这种大的散焦将CTF的第一个零点移动到低得多的空间频率，因此，由于第一个零点之后的相位反转，所获取的层析图像没有超过约3-4 nm分辨率的直接可用信息。CTF校正是实现低温层析成像中2-3 nm分辨率的几种互补方法之一。 我们已经开发了一个图形用户界面程序，它可以稳健地确定倾斜序列中的一个子集的中心散焦(因此CTF的第一个零)。该方法利用周期图平均的方法计算图像的一维功率谱，然后减去显微镜和CCD摄像机的量子噪声功率谱。为了提高信噪比，将不同Z高度条带的功率谱与倾斜图像中心的功率谱进行了近似配准。用户交互地设置在第一个零之前和之后拟合曲线的限制，然后当通过倾斜角度范围分析不同的视图子集时可以应用这些限制。实验表明，在我们的F20和F30显微镜上，计算的散焦与SerialEM程序报告的散焦很好地一致。 在确定中心CTF的第一个零点后，编写第二个程序来反转相位。该校正方法考虑了沿垂直于倾斜轴的方向的离焦梯度。它将图像细分为平行于倾斜轴的条带，因此与散焦相比，条带内的散焦变化较小。然后，假设从倾斜几何体计算出的散焦不变，则对条带进行校正。 在这个阶段，我们仍然不能量化CTF校正带来的分辨率增益。我们的计划是首先使用一些已知晶体蛋白质的图像来验证我们的校正算法，然后将其应用到我们的断层图像中，看看它可以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.