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Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Arnold Stefan A, Albiez Stefan, Opara Nadia, Chami Mohamed, Schmidli Claudio, Bieri Andrej, Padeste Celestino, Stahlberg Henning, Braun Thomas,
Project Fast protein-complex isolation, sample preparation and data processing for high-resolution structural analysis and visual proteomics
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Original article (peer-reviewed)

Journal ACS nano
Volume (Issue) 10(5)
Page(s) 4981 - 8
Title of proceedings ACS nano
DOI 10.1021/acsnano.6b01328

Open Access

URL http://pubs.acs.org/doi/abs/10.1021/acsnano.6b01328?src=recsys
Type of Open Access Publisher (Gold Open Access)

Abstract

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.
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