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Electron Tomography of HEK293T Cells Using Scanning Electron Microscope–Based Scanning Transmission Electron Microscopy

Published online by Cambridge University Press:  02 October 2012

Yun-Wen You ,
Hsun-Yun Chang ,
Hua-Yang Liao ,
Wei-Lun Kao ,
Guo-Ji Yen ,
Chi-Jen Chang ,
Meng-Hung Tsai  and
Jing-Jong Shyue
Yun-Wen You
Affiliation:
Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan
Hsun-Yun Chang
Affiliation:
Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan
Hua-Yang Liao
Affiliation:
Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan
Wei-Lun Kao
Affiliation:
Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan
Guo-Ji Yen
Affiliation:
Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan
Chi-Jen Chang
Affiliation:
Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan
Meng-Hung Tsai
Affiliation:
Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan
Jing-Jong Shyue*
Affiliation:
Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan
*
*Corresponding author. E-mail: shyue@gate.sinica.edu.tw
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Abstract

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

Keywords

electron tomographyscanning transmission electron microscopyscanning electron microscopyHEK293T
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1037 - 1042
Copyright
Copyright © Microscopy Society of America 2012

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