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

Area of Science:

• Electron Microscopy
• Structural Biology
• Image Processing

Background:

• Accurate Transmission Electron Microscopy (TEM) tomography relies on precise alignment of image tilt-stacks.
• Gold nanobeads are commonly used as fiducial markers for determining image rotation axes, but their distribution can be poor or they may be absent.
• The rotational tilt angle is a critical parameter for high-quality tomographic reconstruction, yet its precise determination is often overlooked.

Purpose of the Study:

• To present a straightforward method for determining the rotational tilt angle in TEM tomographic reconstructions.
• To provide a tool that enhances tomogram quality by accurately calculating the rotation axis offset angle.
• To offer a solution for image alignment in TEM tomography, applicable with or without fiducial markers.

Main Methods:

• Utilized the Fast Fourier Transformation (FFT) property to calculate the rotational tilt angle.
• Employed publicly available software packages, IMOD and ImageJ, for the analysis.
• Applied the method to diverse biological samples, including cryo-samples with and without fiducials.

Main Results:

• Successfully determined the rotational tilt angle using FFT, independent of fiducial marker visibility or distribution.
• Reconstructed tomograms exhibited improved quality, notably lacking the characteristic "arc" distortion in cross-sections.
• Demonstrated the tool's versatility on both cryo-samples and embedded thin sections.

Conclusions:

• The FFT-based tool provides a reliable and accessible method for determining the rotational tilt angle in TEM tomography.
• This technique significantly improves tomogram reconstruction accuracy and quality, particularly in challenging cases lacking adequate fiducial markers.
• The developed tool can be readily integrated into existing TEM image processing workflows for enhanced alignment and reconstruction.