title: Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.
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.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Arnold
authors.forename: Stefan
authors.initials: S
authors.lastname: Albiez
authors.affiliation: Paul Scherrer Institute (PSI) , 5232 Villigen, Switzerland.
authors.forename: Nadia
authors.initials: N
authors.lastname: Opara
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Claudio
authors.initials: C
authors.lastname: Schmidli
authors.forename: Andrej
authors.initials: A
authors.lastname: Bieri
authors.affiliation: Paul Scherrer Institute (PSI) , 5232 Villigen, Switzerland.
authors.forename: Celestino
authors.initials: C
authors.lastname: Padeste
authors.forename: Henning
authors.initials: H
authors.lastname: Stahlberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Braun
keywords: electron diffraction; electron microscopy; nanocrystal; negative staining; single-cell analysis; visual proteomics