Tilt-series and electron microscope alignment for the correction of the non-perpendicularity of beam and tilt-axis.
In electron tomography the sample is tilted in the electron microscope and projections are recorded at different viewing angles. In the correct geometric setting, the tilt-axis of the object under scrutiny is perpendicular to the beam direction. However, we will demonstrate that this does not necessarily apply to all electron microscopes equipped with the default column alignment. The resulting effect is that a conical tilt is performed, which has to be considered in the reconstruction to avoid artifacts and to improve the resolution. A novel solution, with significantly improved convergence properties, will be introduced for calculating the three-dimensional marker model, which is necessary for the alignment of the tilt-series. Thereby, the angle between the beam direction and the tilt-axis is calculated, together with other geometrical distortions, like magnification and rotation changes, and incorporated in the reconstruction. Hereby, artifacts can be eliminated at the image processing basis, and the resolution can be significantly improved at the medium to high range frequencies. Synthetical and real data are used to demonstrate the obstructions caused by this effect and the quality improvement of the reconstructions. Finally, we also present a way to align the hardware of the microscope to correct for the non-perpendicularity between the beam direction and the tilt-axis, which is specifically tailored for tomographic applications.
Díez
Daniel Castaño
DC
European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany. castano@embl.de
Seybert
Anja
A
Frangakis
Achilleas S
AS
D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods* / Q000706:statistics & numerical data; D014054:Tomography / Q000379:methods / Q000706:statistics & numerical data