Elferich Johannes, Kong Lingli, Zottig Ximena, Grigorieff Nikolaus
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States.
Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, United States.
Elife. 2024 Dec 20;13:RP97227. doi: 10.7554/eLife.97227.
Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the TEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the TEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm.
透射电子显微镜拍摄的图像通常会受到透镜像差和图像散焦等因素的影响。这些畸变可以在倒易空间中使用对比度传递函数（CTF）进行建模。准确估计和校正CTF对于在低温电子显微镜（cryoEM）中恢复高分辨率信号至关重要。此前，我们在TEM软件包中描述了用于此任务的算法实现（Grant等人，2018年）。在这里，我们表明考虑样品特性，如厚度和倾斜度，可以改善CTF估计。在对细胞样品成像时，这一点尤为重要，因为从准确的Thon环图案建模中得出的样品厚度和几何形状的测量有助于判断样品的质量。这种改进的CTF估计已在TEM程序CTFFIND的新版本CTFFIND5中实现。我们使用倾斜的水通道蛋白晶体和通过聚焦离子束铣削变薄的真核细胞的图像评估了这些估计的准确性。我们估计，对于质量足够好的显微照片，CTFFIND5可以以3°的精度测量样品倾斜度，以5nm的精度测量样品厚度。
