Downing Kenneth H, Glaeser Robert M
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Ultramicroscopy. 2008 Aug;108(9):921-8. doi: 10.1016/j.ultramic.2008.03.004. Epub 2008 Apr 3.
Relatively large values of objective-lens defocus must normally be used to produce detectable levels of image contrast for unstained biological specimens, which are generally weak phase objects. As a result, a subsequent restoration operation must be used to correct for oscillations in the contrast transfer function (CTF) at higher resolution. Currently used methods of CTF correction assume the ideal case in which Friedel mates in the scattered wave have contributed pairs of Fourier components that overlap with one another in the image plane. This "ideal" situation may be only poorly satisfied, or not satisfied at all, as the particle size gets smaller, the defocus value gets larger, and the resolution gets higher. We have therefore investigated whether currently used methods of CTF correction are also effective in restoring the single-sideband image information that becomes displaced (delocalized) by half (or more) the diameter of a particle of finite size. Computer simulations are used to show that restoration either by "phase flipping" or by multiplying by the CTF recovers only about half of the delocalized information. The other half of the delocalized information goes into a doubly defocused "twin" image of the type produced during optical reconstruction of an in-line hologram. Restoration with a Wiener filter is effective in recovering the delocalized information only when the signal-to-noise ratio (S/N) is orders of magnitude higher than that which exists in low-dose images of biological specimens, in which case the Wiener filter approaches division by the CTF (i.e. the formal inverse). For realistic values of the S/N, however, the "twin image" problem seen with a Wiener filter is very similar to that seen when either phase flipping or multiplying by the CTF is used for restoration. The results of these simulations suggest that CTF correction is a poor alternative to using a Zernike-type phase plate when imaging biological specimens, in which case the images can be recorded in a close-to-focus condition, and delocalization of high-resolution information is thus minimized.
对于未染色的生物标本（通常是弱相位物体），通常必须使用相对较大的物镜散焦值来产生可检测水平的图像对比度。因此，必须使用后续的恢复操作来校正高分辨率下对比度传递函数（CTF）中的振荡。当前使用的CTF校正方法假设了一种理想情况，即散射波中的弗里德尔对在图像平面中贡献了相互重叠的傅里叶分量对。随着颗粒尺寸变小、散焦值变大和分辨率变高，这种“理想”情况可能只能得到很差的满足，或者根本无法满足。因此，我们研究了当前使用的CTF校正方法在恢复因有限尺寸颗粒直径的一半（或更多）而发生位移（离域）的单边带图像信息方面是否也有效。计算机模拟表明，通过“相位翻转”或乘以CTF进行恢复只能恢复大约一半的离域信息。另一半离域信息进入在线全息图光学重建过程中产生的那种双散焦“孪生”图像。只有当信噪比（S/N）比生物标本低剂量图像中的信噪比高几个数量级时，使用维纳滤波器进行恢复才能有效地恢复离域信息，在这种情况下，维纳滤波器接近除以CTF（即形式上的逆）。然而，对于实际的S/N值，使用维纳滤波器时出现的“孪生图像”问题与使用相位翻转或乘以CTF进行恢复时看到的问题非常相似。这些模拟结果表明，在对生物标本成像时，CTF校正相对于使用泽尼克型相位板是一种较差的选择，在使用相位板的情况下，可以在接近聚焦的条件下记录图像，从而将高分辨率信息的离域最小化。
