Han K F, Sedat J W, Agard D A
Department of Biochemistry and Biophysics, University of California at San Francisco 94143-0448, USA.
J Microsc. 1995 May;178(Pt 2):107-19. doi: 10.1111/j.1365-2818.1995.tb03586.x.
With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0.3-1.0 microns). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than approximately 10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the 'restored' images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.
随着频率的增加，人们正在使用厚切片（0.3 - 1.0微米）的电子显微镜断层扫描技术以高分辨率研究细胞器和核结构。为了从观察到的图像强度准确地三维重建结构，理解图像强度与标本质量密度之间的关系至关重要。厚标本成像因多重散射的很大一部分而变得复杂，这会产生非相干和部分相干的图像成分。在这里，我们研究了200 keV和300 keV下厚生物标本的成像机制，以便从非相干（多重散射）成分中分辨出相干散射成分。使用了两种技术：电子能量损失光谱成像（ESI）和使用聚焦系列的出射波前重建。尽管通常认为厚标本的成像主要由振幅（吸收）对比度主导，但我们发现对于传统染色的生物标本，相位对比度有显著贡献，并且在分辨率优于约10 nm时，叠加的相位对比度占主导。结果表明，相干散射随标本厚度的降低与多重散射的增加直接相关。进一步表明，出射波前重建可以在成像过程中排除显微镜像差以及多重散射成分。由于这些厚标本的大多数非弹性散射实际上是多重非弹性散射，因此证明出射波前重建可以起到部分能量过滤的作用。通过排除多重散射，“恢复”的图像显示出增强的对比度和分辨率。这些发现对于厚生物标本的三维重建具有直接影响，在三维重建中，人们曾假设图像强度与质量密度之间存在简单的直接关系，并且像差未得到校正。
