Turner J N, Rieder C L, Collins D N, Chang B B
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
J Electron Microsc Tech. 1989 Jan;11(1):33-40. doi: 10.1002/jemt.1060110106.
Optimal imaging of complex structures requires proper alignment relative to the optic axis of the electron microscope. This is especially important for high-voltage and intermediate-voltage microscopes, which form an in-focus image throughout the entire thickness of the object. As a result, structures at different specimen heights form overlapping and confused images that severely curtail the usefulness of these instruments. The work described here provides a generalized, flexible method for optimizing specimen orientation and eliminating or limiting image overlap by means of a commonly used double-tilt stage. Analysis of the motion about the two axes provides accurate tilting for any azimuthal direction whether or not it corresponds to a mechanical axis of the stage. An object can be positioned to minimize image overlap, to record stereopairs for any parallax axis, and to record three-dimensional data sets by the conical collection geometry. Images of muscle paracrystals are shown after tilting about an axis perpendicular to a symmetry direction. The tilted image displays higher-order symmetry, which is altered by changes of one degree. Precision double-tilting for optimizing stereopairs is shown for a desmosome recorded using different parallax axes and pretilts. A tomographic conical data-collection scheme is demonstrated by imaging a microtubule axoneme for a specific cone half-angle and arbitrary azimuthal angles.
对复杂结构进行最佳成像需要相对于电子显微镜的光轴进行适当的对齐。这对于高压和中压显微镜尤为重要，因为它们在物体的整个厚度范围内形成聚焦图像。结果，不同标本高度的结构会形成重叠且模糊的图像，这严重限制了这些仪器的实用性。本文所述的工作提供了一种通用、灵活的方法，可通过常用的双倾台来优化标本取向并消除或限制图像重叠。对围绕两个轴的运动进行分析可为任何方位方向提供精确的倾斜，无论该方向是否对应于倾台的机械轴。可以对物体进行定位，以最小化图像重叠、为任何视差轴记录立体对，并通过锥形采集几何结构记录三维数据集。展示了在垂直于对称方向的轴上倾斜后肌肉副晶体的图像。倾斜图像显示出更高阶的对称性，角度变化一度就会改变这种对称性。对于使用不同视差轴和预倾斜记录的桥粒，展示了用于优化立体对的精确双倾斜。通过对特定锥半角和任意方位角的微管轴丝进行成像，演示了断层扫描锥形数据采集方案。
