Fiducial-less alignment of cryo-sections.
Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.
Castaño-Díez
Daniel
D
European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany. Castano@embl.de
Al-Amoudi
Ashraf
A
Glynn
Anne-Marie
AM
Seybert
Anja
A
Frangakis
Achilleas S
AS
D000465:Algorithms; D053000:Analytic Sample Preparation Methods / Q000592:standards*; D016616:Cryoultramicrotomy / Q000592:standards*; D021621:Imaging, Three-Dimensional; D012984:Software