Lipid nanotube encapsulating method for two- and three-dimensional transmission electron microscopy analyses of cage-shaped proteins

A novel, sample-fixation technique for transmission electron microscopy (TEM) is proposed. We encapsulated various samples in one-dimensional hollow cylinders of a glycolipid nanotube (LNT) and found it to be highly stable, flexible, and reproducible. The cylindrical shape of the LNT allows us to observe nanomaterials consistently even when the samples are rotated around the tube axis. This is critical in improving TEM computed tomography (CT), which suffers from the loss of the electron beam at high tilting angles because of the increased effective thickness of the supporting film. This technique makes it possible to reconstruct three-dimensional images from weak-signal TEM images. In this work, we examined two types of cage-shaped proteins, ferritin, and DNA-binding protein from starved cells (Dps). We reconstructed three-dimensional images of Fe cores from zero-loss images or from electron energy-loss spectroscopy (EELS) mapping images obtained with the Fe M(2.3) edge. We also applied negative staining and vitreous ice embedding (VIE). These results show the potential of the LNT encapsulation technique.