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Updated: Jun 8, 2026

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

Published on: March 12, 2017

The orthogonal tilt reconstruction method.

Andres Leschziner¹

¹Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

Methods in Enzymology

|October 5, 2010

Summary

Random Conical Tilt (RCT) and Orthogonal Tilt Reconstruction (OTR) are cryo-EM methods for modeling asymmetric molecules. OTR offers fully sampled structures, overcoming RCT

Area of Science:

Cryo-electron microscopy
Structural biology
Biophysics

Background:

Generating initial models for novel asymmetric molecules in cryo-electron microscopy (cryo-EM) is challenging.
Geometric reconstruction methods, like Random Conical Tilt (RCT), utilize tilted microscope views but suffer from incomplete Fourier space sampling.
Orthogonal Tilt Reconstruction (OTR) offers a solution for fully sampled structures.

Purpose of the Study:

To review and compare the performance of Random Conical Tilt (RCT) and Orthogonal Tilt Reconstruction (OTR) methods.
To discuss the practical implementation steps for Orthogonal Tilt Reconstruction (OTR).
To evaluate these geometric reconstruction techniques on a biological test sample.

Main Methods:

Data collection using Random Conical Tilt (RCT) with images at high tilt (~50°) and 0°.

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Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

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Data collection using Orthogonal Tilt Reconstruction (OTR) with images at -45° and +45° tilt.

Comparison of reconstruction quality and sampling completeness between RCT and OTR.

Main Results:

RCT provides reconstructions independent of sample orientation but yields incomplete Fourier space sampling.
OTR, when applied to samples with diverse orientations, achieves fully sampled structures.
Performance comparison on a biological sample highlights OTR's advantages in data completeness.

Conclusions:

Orthogonal Tilt Reconstruction (OTR) is a valuable advancement over Random Conical Tilt (RCT) for achieving complete structural models in cryo-EM.
OTR's requirement for a large number of sample orientations is a key consideration for its application.
The study provides insights into the practical application and comparative performance of these geometric reconstruction techniques.