CONTRAST TRANSFER FUNCTION (CTF) CORRECTION FOR ELECTRON TOMOGRAPHY

电子断层扫描的对比度传递函数 (CTF) 校正

• 批准号: 7955054
• 负责人: DAVID N MASTRONARDE
• 金额: $ 1.61万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955054
• 关键词: Accounting; Algorithms; Cellular Structures; Computer Retrieval of Information on Scientific Projects Database; Dose; Frequencies; Funding; Grant; Height; Image; Institution; Methods; Microscope; Noise; Optics; Phase; Proteins; Reporting; Research; Research Personnel; Resolution; Resources; Series; Signal Transduction; Source; Staging; Tomogram; United States National Institutes of Health; Variant; Writing; charge coupled device camera; electron tomography; improved; particle; programs; quantum; research study; Accounting; Algorithms; Cellular Structures; Computer Retrieval of Information on Scientific Projects Database; Dose; Frequencies; Funding; Grant; Height; Image; Institution; Methods; Microscope; Noise; Optics; Phase; Proteins; Reporting; Research; Research Personnel; Resolution; Resources; Series; Signal Transduction; Source; Staging; Tomogram; United States National Institutes of Health; Variant; Writing; charge coupled device camera; electron tomography; improved; particle; programs; quantum; research study

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 使用常规光学器件（即，在没有相板的情况下），低剂量冷冻-ET中的图像对比度主要是通过显着偏置显微镜（通常为3-8微米）来实现。 这个大的散焦将CTF的第一个零移到了较低的空间频率，因此，由于第一个零以后的相位反转，获得的断层图无法直接具有可用信息。 CTF校正是在冷冻图中实现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，该程序可以坚固地确定中心散焦（因此是CTF的第一个零），以从倾斜系列中的视图子集。 我们的方法使用定期图平均计算图像的1D功率谱，然后从中减去显微镜和CCD摄像头的量子噪声功率谱。 为了提高信号与噪声比，不同Z高度的条带的功率光谱与倾斜图像中心的近似配准转移到近似的注册中。 用户在第一个零之前和之后交互式设置拟合曲线的限制，然后在通过倾斜角范围分析视图子集时可以应用这些限制。 实验表明，计算出的散焦与Serialem程序在我们的F20和F30显微镜上报告的散焦吻合。 编写第二个程序以逆转CTF中心中的第一个零之后，以逆转相位。 校正方法考虑了沿垂直于倾斜轴的方向的散焦梯度。 它将图像插入平行于倾斜轴的条中，以使带中的散焦变化与散焦相比很小。 然后，它纠正了带有从倾斜几何形状计算的恒定散热器的条带。 在此阶段，我们仍然无法量化由于CTF校正而引起的分辨率增益。该计划是首先使用来自某些已知晶体蛋白的图像验证我们的校正算法，然后将其应用于我们的断层图，以查看它可以改善粒子平均程序的最终结果。