Koeck P J B, Karshikoff A
Royal Institute of Technology, School of Technology and Health, Handen, Sweden.
Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
J Microsc. 2015 Sep;259(3):197-209. doi: 10.1111/jmi.12253. Epub 2015 Apr 28.
We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors.
我们建立了通过透射电子显微镜对薄标本成像时，相位以及相位和振幅物体描述的级数展开式中的线性项和二次项的表达式。基于这些表达式，我们模拟了不同厚度的未染色蛋白质复合物图像的相应贡献，并得出了每个项对图像对比度贡献的估计值。由此我们可以估计出弱相位、弱振幅和相位物体近似（以及因此的线性成像）仍然相当准确的最大标本厚度。在讨论厚标本时，由于将图像描述为标本的滤波投影，还需要考虑其局限性，因为标本的不同层并非以相同的散焦值成像。因此，我们将基于投影近似的模拟与更精确的多切片成像模型进行了比较。然而，我们发现，对于模拟中选择的散焦值，非线性图像贡献导致的误差大于散焦梯度导致的误差。最后，我们研究了所讨论的非线性图像贡献和散焦梯度如何影响三维重建的质量。我们发现，三维重建在达到高分辨率的同时会呈现局部系统性结构误差。
