Optimum specimen positioning in the electron microscope using a double-tilt stage

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.