Taking pictures of single proteins is an important goal in biology and medicine, allowing the study of protein folding and other behavior, and possibly leading to new approaches in drug development. Imaging single proteins, however, requires high-intensity electron beams or x-ray sources, which necessarily damage the protein. An alternative is to use low-intensity radiation for imaging, however then the exposure time has to increase and the proteins tend to drift away from the photo. Now Jean-Nicolas Longchamp and colleagues at the University of Zurich in Switzerland have used Graphenea’s CVD graphene as a transparent trap that holds proteins in place while they’re imaged with a low-energy electron beam. We caught up with Jean-Nicolas for a few words about the result.

1) How does your protein imaging technique work?

Our imaging technique is called low-energy electron holography. In the experiment, a sharp metal tip acts as a source of highly coherent electrons. The atomic sized electron emitter is brought as close as 100nm to the sample with nanopositioning. Part of the electron wave is elastically scattered off the object and hence is called the object wave, while the un-scattered part of the wave represents the reference wave. At a distant detector, the hologram, i.e. the pattern resulting from the interference of these two wave fronts is recorded. A hologram, in contrast to a diffraction pattern, contains the phase information of the object wave, and the object structure can thus be reconstructed easily and unambiguously.

2) How does graphene help the imaging?

In order to enable the imaging, the proteins need to be deposited onto a transparent and conductive substrate for low-energy electrons. We could show that ultraclean freestanding graphene is highly transparent (more than 70%) for those electrons. To attain such high transparency, the graphene samples were prepared by our patented method based on the catalytic activity of Pt-metal. While until today, solely graphene has been demonstrated to be a suitable substrate for low-energy electron holography, we are trying to prepare other 2D materials (as for instance h-BN) and 2D-heterostructures in an ultraclean freestanding manner to investigate their properties.

3) What's the significance of the particular proteins you used in this study? What's the immediate medical application?

Currently, we are in the process of developing our imaging technique for structural biology purposes, with the goal of imaging individual proteins at atomic resolution. For that, we are now imaging proteins whose structures are already known from X-ray crystallography investigations and compare our images to the proposed atomic models. We are sure that once we have attained atomic resolution, the pharmaceutical industry would be very interested in images of proteins with unknown structures, in particular proteins which could not be crystallized. Images of individual proteins at atomic resolution will deliver very important information for the design of novel and better drugs.

4) How does your method fare against other protein imaging techniques?

Most of the protein structural information available today has been obtained from either X-ray crystallography experiments or cryo-electron microscopy investigations by means of averaging over many molecules assembled into a crystal or over a large ensemble selected from low signal-to-noise ratio electron micrographs respectively. Despite the impressive amount of available data, a strong desire for acquiring structural data from just one individual molecule is emerging for good reasons. Most of the biologically relevant molecules exhibit different conformations; the associated structural details however, remain undiscovered when averaging is involved. Moreover, a large subset of the entirety of proteins, in particular out of the important category of membrane proteins, does not crystallize at all. If just one individual protein or protein complex can be analyzed in sufficient detail, also those objects become accessible.

For a meaningful contribution to structural biology, a tool for single molecule imaging has to allow for observing an individual protein long enough to acquire a sufficient amount of data for revealing its structure, ideally without destroying it. The strong inelastic scattering cross-section for both X-rays and high-energy electrons as employed in state-of-the-art aberration corrected TEMs, inhibits accumulation of sufficient elastic scattering events required in order to reveal high-resolution reconstruction of just one molecule. Future X-ray Free Electron Lasers (FELs) with drastically enhanced brightness and reduced pulse duration might eventually achieve the goal of single molecule imaging. Yet, the current and foreseeable state-of-the-art in FEL performance still requires averaging over at least 1 million molecules.

In contrast to this, biomolecules as for instance DNA or proteins withstand irradiation by low-energy electrons and remain unperturbed even after a total dose of at least 5 orders of magnitude larger than the permissible dose in X-ray or high-energy electron imaging. The damage-free radiation of low-energy electrons combined with the fact that the de Broglie wavelengths associated with this energy range are of the order of 1Å, make low-energy electron holography an auspicious candidate for structural biology at the single molecule level.

5) What's next for this project?

The next goal for this project is to attain atomic resolution imaging of individual proteins. For that, a low-energy electron holographic microscope capable of operation at cryogenic temperatures while keeping very high mechanical stability, which is mandatory for holography, needs to be designed and implemented.

Thank you Jean-Nicolas for your time.