Connecting μ-fluidics to electron microscopy

Abstract

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.

Introduction

Systems biology aims to quantify the molecular elements of a biological system, to determine their interactions and to integrate this information into network models (Aderem, 2005). The development of comprehensive models requires experimental information about the spatial and temporal arrangements of the network components as well as their structure, a challenge that required a multi-resolution approach and the combination of different techniques (Aloy and Russell, 2006, Kherlopian et al., 2008). Cryo-electron tomography (cryo-ET) is the ultimate technique to reveal the spatial organisation of protein structures and macromolecular complexes in single cells (Ben-Harush et al., 2010, Lučić et al., 2005). Currently cryo-ET is restrained by several limitations, such as the size of the cell that can be analysed (maximum diameter of ∼2 μm) (Leis et al., 2009), and by problems in data segmentation and in the template matching required for protein recognition (restricted to relatively large protein complexes) (Bohm et al., 2000). Indeed, many target structures can only be recognised if labelled with electron-dense markers (e.g., gold labels) (Nickell et al., 2006), which, despite recent progress (Kireev et al., 2008), often involves harsh preparative treatment of the cells. Furthermore, while ET delivers structural and spatial information, correlation of these with other methods such as mass spectroscopy (MS) (Aebersold and Mann, 2003) is difficult. A complementary approach is to physically lyse the cells and to subsequently write the entire sample onto electron microscopy (EM) grids for structure analysis by transmission EM (TEM), or mass analysis by scanning TEM (STEM). Ultimately, the use of microfluidic techniques offers the potential to analyse a single cell by making it possible to investigate protein ultrastructures and membrane fragments in lysates (Engel, 2010).

Here we present a lossless sample deposition method for EM (Fig. 1), which allows the handling of minute sample volumes and immobilisation of the total sample content on EM grids to obtain, for example, the full cell inventory. This methodology is combined with a microfluidic sample-conditioning module. The constellation provides a new staining technique for heavy metal salts (negative stain) for TEM as well as specific desalting for mass measurements by STEM.