Technical note: A tool for determining rotational tilt axis with or without fiducial markers

Virtually all reliable TEM tomographic reconstructions in life science depend upon cross-correlating successive images in a tilt-stack, and then using gold nanobeads as fiducial markers for determining the relative image rotation axis. Although the rotational tilt angle is one of the essential parameters affecting the quality of tomographic reconstructions, little is discussed about its determination. We provide here a simple tool based on the property of Fast Fourier Transformation for determining this rotation axis offset angle. Our method uses two publicly-available software packages (IMOD and ImageJ), and can be used on any TEM-based image stack, which is useful for images with poor bead distribution or situations where such beads are not visible. We illustrate this procedure with two different biological samples, one of which is a plunge frozen cryo-sample with fiducials and the other an epon-embedded thin section without fiducials. Prior knowledge of the rotational tilt angle facilitates further processing tomograms. With cross-correlation and the FFT-obtained rotational tilt angle, we reconstructed tomograms, of which the cross-section did not show “arc” distortion. This tool could be easily incorporated into any software for the alignment with or without fiducials.