Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box
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- 作者：Jason Pierson ; José ; Jesú ; s Ferná ; ndez ; Erik Bos ; Shoaib Amini ; Helmut Gnaegi ; Matthijn Vos ; Bennie Bel ; Freek Adolfsen ; José ; L. Carrascosa ; Peter J. Peters
- 关键词：Anti-contamination glove box ; Electrostatic charging ; Vitreous cryo-sections ; Cryo-electron tomography ; 80S ribosome
- 刊名：Journal of Structural Biology
- 出版年：2010
- 出版时间：February 2010
- 年：2010
- 卷：169
- 期：2
- 页码：219-225
- 全文大小：577 K
文摘
Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.
Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae.