Fiducial Marker and Hybrid Alignment Methods for Single- and Double-axis Tomography

  • David N. Mastronarde


Accurate alignment of projection images is an important step in obtaining a high-quality tomographic reconstruction. Ideally, all images should be aligned so that each represents a projection of the same 3D object at a known projection angle. Inadequate image alignment will result in blurring or smearing of features in the reconstruction. The problem is made more difficult because exposure to the electron beam during the acquisition of a tilt series induces geometric changes in many samples (primarily plastic-embedded material). For any sample, but particularly for ones that are not rigid during imaging, a powerful method of image alignment uses the measured coordinates of fiducial markers through the series of images; these can be fit to equations that describe the image projection. Alternative methods are based on cross-correlation of images. The fiducial marker method has the advantage that it guarantees a consistent alignment among the images from the full range of tilt angles. It is also more easily adapted to correct for changes in the sample that occur during imaging.


Tilt Angle Gold Particle Local Alignment Projection Image Fiducial Marker 


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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • David N. Mastronarde
    • 1
  1. 1.The Boulder Laboratory for 3D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental BiologyUniversity of ColoradoBoulderUSA

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