Graphene: Substrate preparation and introduction

Abstract

This technical note describes the transfer of continuous, single-layer, pristine graphene to standard Quantifoil TEM grids. We compare the transmission properties of pristine graphene substrates to those of graphene oxide and thin amorphous carbon substrates. Positively stained DNA imaged across amorphous carbon is typically indiscernible and requires metal shadowing for sufficient contrast. However, in a practical illustration of the new substrates properties, positively stained DNA is imaged across pristine graphene in striking contrast without the need of metal shadowing. We go onto discuss technical considerations and the potential applications of pristine graphene substrates as well as their ongoing development.

Introduction

Despite the benefits of highly transparent crystalline substrates being long since recognized, technical difficulties with their preparation have prevented wide scale application (Dobelle and Beer, 1968, Hahn and Baumeister, 1974). Recent developments in the large scale synthesis of pristine graphene (Li et al., 2009c) present interesting possibilities for structural techniques that up until now have required amorphous carbon substrates (e.g. 2D electron crystallography and new emerging methods (Benesch et al., submitted for publication, Kelly et al., 2008, Rhinow and Kühlbrandt, 2008)).

Crystalline substrates are effectively transparent to transmission electron microscopy (TEM) at resolutions below their periodicity, and at higher resolutions the periodic nature of the signal facilitates subtraction if necessary (Meyer et al., 2008a). At crystalline periodicities of 2.13 and 1.23 Å, respectively (Meyer et al., 2007), structural details of pristine graphene are outside the resolutions typically resolved in biological TEM. At a single layer thickness of 0.34 nm (Eda et al., 2008), the minimal scattering cross-section of pristine graphene also minimizes background (noise) contributed by inelastic and multiple scattering within the substrate. Other remarkable properties are also derived from the highly ordered structure of graphene, including high mechanical strength/elasticity (Lee et al., 2008, Wang et al., 2009a, Zakharchenko et al., 2009) and “ballistic” electrical conductivity, also at liquid nitrogen temperatures (Heersche et al., 2007, Zhang et al., 2005). Although the threshold for knock-on damage is ∼86 keV (Zobelli et al., 2007), we have found graphene substrates to be remarkably stable, withstanding acceleration voltages of up to 300 keV under the typically lower electron dose conditions (as little as 20–30 e/Ų) of biological TEM. The electrical conductivity of graphene, converted into bulk units and assuming a thickness of 3.4 Å, is more than six orders of magnitude higher than that of amorphous carbon (Chen et al., 2008, Robertson, 1986, Ziegler, 2006). Hence, graphene substrates may potentially reduce the effects of charging and improve the imaging stability of insulating materials like amorphous ice.

In previous work we introduced the use of graphene oxide (G-O), a hydrophilic derivative of pristine graphene with semi-crystalline properties (Pantelic et al., 2010). Surface bound, oxidized functional groups contribute to the hydrophilic properties of the substrate, but also introduce a weak background signal. Oxidization also increases the thickness of pristine graphene to ∼1 nm (Stankovich et al., 2006, Wang et al., 2009b), consequently increasing inelastic scattering within the substrate and introducing additional background noise. But in particular, deposition from solution produces substrates composed of overlapped/stacked platelets that are thus inhomogeneous.

Chemical vapor deposition (CVD) is a method of chemically producing continuous areas of pristine monolayer (>95%, and recently completely monolayer (Li et al., 2009a, Li et al., 2009b)) graphene across thin Cu foils of any size (Li et al., 2009c, Yu et al., 2008). From these Cu foils, the graphene is directly transferrable to standard Quantifoil TEM grids by evaporation of solvent (to adhere the graphene) followed by chemical wet etching (to dissolve the Cu foil). This technical note discusses a method by which graphene is transferred and compares the substrate to previous graphene oxide and amorphous carbon substrates. The high transparency of the substrate is illustrated by an example in which positively stained double-stranded DNA is imaged in high contrast, without the necessity of metal shadowing.