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A Novel Method of Data Collection for Automated Electron Tomography Based upon Pre-cal1bration of Image Shifts and Defocus Changes

Published online by Cambridge University Press:  02 July 2020

Ulrike Ziese ,
Ries Janssen ,
Willie Geerts ,
Theo van der Krift ,
Auke van Balen ,
Hans de Ruijter ,
Krijn de Jong ,
Arie Verkleij  and
Bram Koster
Ulrike Ziese
Affiliation:
Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands
Ries Janssen
Affiliation:
Inorganic Chemistry and Catalysis, Utrecht University, 3584 CA, Utrecht, The Netherlands;
Willie Geerts
Affiliation:
Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands
Theo van der Krift
Affiliation:
Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands
Auke van Balen
Affiliation:
FEI / Philips Electron Optics, 5600 MD, Eindhoven, The Netherlands;
Hans de Ruijter
Affiliation:
Emispec Systems Inc., Tempe, AZ, 85282, USA
Krijn de Jong
Affiliation:
Inorganic Chemistry and Catalysis, Utrecht University, 3584 CA, Utrecht, The Netherlands;
Arie Verkleij
Affiliation:
Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands
Bram Koster
Affiliation:
Molecular Cell Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands
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Abstract

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments. For the 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide 3D information with nm-scale resolution of individual and unique samples. Routine application of electron tomography will comprise a major step forward in the characterization of complex materials and cellular arrangements. When collecting tilt series for electron tomography image shifts and defocus changes have to be corrected for by the human operator. The repetitive correction of these changes is highly time consuming, error prone and very hard to carry out under low-dose imaging conditions.

Many practical problems are overcome when electron tomography data collection is performed in an automated fashion. Automation includes the (a) image acquisition on a (digital) CCD camera, which implies that (b) changes in image position and defocus can be detected by on-line image processing and (c) immediately be corrected for by computer control of the microscope, (d) Finally, tilt series are directly available in digital format for subsequent processing. Typically, carrying out such an experiment would take a day, and the actual data collection 2-4 hours. in spite of the enormous progress made in terms of data collection speed during the last few years, the current status of automated tomography still does not meet the requirements that would make it a routinely applicable tool. For a great number of biological assays and research projects, results obtained under different experimental conditions have to be compared, and series of experiments have to be carried out. Therefore, we propose a novel approach for recording a tilt series that significantly increases data collection speed, and widens the applicability of the technique.

Type
Electron Tomography: Recent Advances and Applications (Organized by M. Marko)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 78 - 79
Copyright
Copyright © Microscopy Society of America 2001

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