Graphene oxide: A substrate for optimizing preparations of frozen-hydrated samples

Abstract

Graphene oxide is a hydrophilic derivative of graphene to which biological macromolecules readily attach, with properties superior to those of amorphous carbon films commonly used in electron microscopy. The single-layered crystalline lattice of carbon is highly electron transparent, and exhibits conductivity higher than amorphous carbon. Hence, graphene oxide is a particularly promising substrate for the examination of biological materials by electron microscopy. In this manuscript we compare graphene oxide films to commonly used amorphous carbon films, describing the use of graphene in optimizing the preparation of unstained, vitrified biological macromolecules.

Introduction

Films made of evaporated carbon are routinely used as substrates for the mounting and subsequent imaging of biological samples by electron microscopy. When imaged at higher magnifications such amorphous carbon substrates display a granular texture, attenuating and even obscuring the signal of unstained particles. Hence, to circumvent this limitation frozen-hydrated samples are often imaged within unsupported regions of vitreous ice prepared across perforated carbon films. However, a problem sometimes encountered with such preparations is the tendency of biological macromolecules to adsorb strongly to the surrounding carbon leading to a depletion of particles within the spanning vitreous ice. This in particular, is an issue with preparations that require washing to remove unwanted low molecular weight constituents, as most of the sample is also subsequently removed.

Graphene is a single-layer crystalline lattice of sp² bound carbon atoms (Geim and Novoselov, 2007) possessing several advantages over amorphous carbon. The ideal (i.e. uncontaminated and defect free) graphene structure is expected to be essentially featureless down to a resolution of 2.13 Å. If sampled at higher resolution, the regular crystallinity of graphene gives rise to a periodic signal that can be subtracted if necessary (Meyer et al., 2008). The single-layer thickness (0.34 nm, Eda et al., 2008) of graphene also minimizes electron scattering within the substrate and hence any background noise. The conductivity of pristine graphene, converted to bulk units assuming a thickness of 3.4 Å, is more than six orders of magnitude higher than the conductivity of amorphous carbon (Chen et al., 2008, Robertson, 1986, Ziegler, 2006). Therefore, graphene may reduce charging effects and improve the imaging stability of insulating materials like amorphous ice. Graphene reacts to deformation elastically (Lee et al., 2008, Zakharchenko et al., 2009) and has a high mechanical strength (Wang et al., 2009), allowing it to withstand sonication as well as other harsh (chemical) treatments (Reina et al., 2009).

Although hydrophobic, graphene can be functionalized using chemical processes (Wang et al., 2009, Schniepp et al., 2006), thereby producing hydrophilic substrates to which molecules readily attach. Graphene oxide is one such derivative produced by the exfoliation of graphite oxide, a heavily oxygenated and hydrophilic form of graphite (Paredes et al., 2008, Wei et al., 2008). Fig. 1 shows an area of 1–2 layer thin graphene oxide substrate. Although oxidization attenuates the material properties of pristine graphene (see Section 4), substrates are stable and demonstrate significantly reduced background contrast. Clearly defined hexagonal diffraction patterns (Fig. 1, insets A and B) occurring at the material periodicities (2.13 and 1.23 Å, respectively, Meyer et al., 2007) correspond to disordered stacking of individual graphene oxide layers.

When preparing difficult and fragile samples, sometimes also requiring multiple washes prior to vitrification, the use of additional amorphous carbon substrate is routine. In this manuscript we compare graphene oxide to amorphous carbon and describe its use as a supporting substrate for unstained, frozen-hydrated samples.