Rapid tilt-series method for cryo-electron tomography: Characterizing stage behavior during FISE acquisition

ABSTRACTWe and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.

Download Full-text

Endocytosed nanogold fiducials for improved in-situ cryo–electron tomography tilt-series alignment

Journal of Structural Biology ◽

10.1016/j.jsb.2021.107698 ◽

2021 ◽

Vol 213 (1) ◽

pp. 107698

Author(s):

Casper Berger ◽

Raimond B.G. Ravelli ◽

Carmen López-Iglesias ◽

Peter J. Peters

Keyword(s):

Electron Tomography ◽

Cryo Electron Tomography ◽

Tilt Series

Download Full-text

Three-Dimensional Reconstruction of Two Forms of the Chaperonin GRO EL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180045 ◽

1990 ◽

Vol 48 (1) ◽

pp. 258-259

Author(s):

J.L. Carrascosa ◽

G. Abella ◽

S. Marco ◽

M. Muyal ◽

J.M. Carazo

Keyword(s):

Three Dimensional ◽

The Other ◽

Two Dimensional ◽

Series Method ◽

Three Dimensional Reconstruction ◽

Structural Components ◽

E Coli ◽

Dimensional Reconstruction ◽

Morphogenetic Factors ◽

Tilt Series

Chaperonins are a class of proteins characterized by their role as morphogenetic factors. They trantsiently interact with the structural components of certain biological aggregates (viruses, enzymes etc), promoting their correct folding, assembly and, eventually transport. The groEL factor from E. coli is a conspicuous member of the chaperonins, as it promotes the assembly and morphogenesis of bacterial oligomers and/viral structures.We have studied groEL-like factors from two different bacteria:E. coli and B.subtilis. These factors share common morphological features , showing two different views: one is 6-fold, while the other shows 7 morphological units. There is also a correlation between the presence of a dominant 6-fold view and the fact of both bacteria been grown at low temperature (32°C), while the 7-fold is the main view at higher temperatures (42°C). As the two-dimensional projections of groEL were difficult to interprete, we studied their three-dimensional reconstruction by the random conical tilt series method from negatively stained particles.

Download Full-text

Embedded Gold Markers for Improved TEM/STEM Tomography Reconstruction

ISTFA 2008: Conference Proceedings from the 34th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa2008p0172 ◽

2008 ◽

Author(s):

Jian-Shing Luo ◽

Chia-Chi Huang ◽

Jeremy D. Russell

Keyword(s):

3D Reconstruction ◽

Feature Tracking ◽

Semiconductor Device ◽

Electron Tomography ◽

Tracking Process ◽

Carbon Coated ◽

Novel Method ◽

Tomography Reconstruction ◽

Tilt Series ◽

20 Nm

Abstract Electron tomography includes four main steps: tomography data acquisition, image processing, 3D reconstruction, and visualization. After acquisition, tilt-series alignments are performed. Two methods are used to align the tilt-series: cross-correlation and feature tracking. Normally, about 10-20 nm of fiducial markers, such as gold beads, are deposited onto one side of 100 mesh carbon-coated grids during the feature-tracking process. This paper presents a novel method for preparing electron tomography samples with gold beads inside to improve the feature tracking process and quality of 3D reconstruction. Results show that the novel electron tomography sample preparation method improves image alignment, which is essential for successful tomography in many contemporary semiconductor device structures.

Download Full-text