Imaging DNA nanostructures with advanced TEM techniques

The low contrast of biomolecules in TEM has been a great obstacle for their structure determination and hence to the understanding of their structure-function relation. Historically, single DNA strands remained one the most difficult classes of biomolecular specimens to image, due to low electron scattering strength of its constituent elements. The common practice was then to image them either when freely suspended (without any support) or shadow image them with negative staining technique. Those remedies are limited in terms of applicability to different DNA nanostructures as well as pose difficulties in sample preparation. For example, making the 2D DNA nanostructures freestanding would not be a viable solution for imaging them. This thesis provides a general study to tackle the challenges in imaging nucleic acids with TEM...BN/Cees Dekker La

Distortion of DNA Origami on Graphene Imaged with Advanced TEM Techniques

While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates.BN/Cees Dekker LabBN/Technici en AnalistenQN/AfdelingsbureauQN/Zandbergen La

Visualization of unstained DNA nanostructures with advanced in-focus phase contrast TEM techniques

Over the last few years, tremendous progress has been made in visualizing biologically important macromolecules using transmission electron microscopy (TEM) and understanding their structure-function relation. Yet, despite the importance of DNA in all forms of life, TEM visualization of individual DNA molecules in its native unlabeled form has remained extremely challenging. Here, we present high-contrast images of unstained single-layer DNA nanostructures that were obtained using advanced in-focus phase contrast TEM techniques. These include sub-Ångstrom low voltage electron microscopy (SALVE), the use of a volta-potential phase plate (VPP), and dark-field (DF) microscopy. We discuss the advantages and drawbacks of these techniques for broad applications in structural biology and materials science.BN/Cees Dekker LabQN/AfdelingsbureauQN/Zandbergen La