Kuniaki Nagayama

Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of approximately 3, compared with conventional cryoEM. The reconstruction was carried out to 13 A resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.

Visualizing virus assembly intermediates inside marine cyanobacteria

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Zernike phase contrast electron microscopy of ice-embedded influenza A virus.

The ultrastructure of the frozen-hydrated influenza A virus was examined by Zernike phase contrast electron microscopy. Using this new microscopy, not only lipid bilayers but also individual glycoprotein spikes on viral envelopes were clearly resolved with high contrast in micrographs taken in focus. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. About 450 glycoprotein spikes were present in an average-sized spherical virion. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished in one viral particle. Thus, Zernike phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables high-contrast images of ice-embedded particles free of contrast transfer function artifacts that can be a problem in conventional cryo-electron microscopy.

Phase Contrast Enhancement with Phase Plates in Electron Microscopy

Publisher Summary This chapter discusses the significant role of phases in transmission electron microscopy (TEM). The transparency of phase objects is recognized as no change in the intensity of waves (the wave magnitude) before and after the object is in the penetration. Central in the image formation of phase objects is the recovery of the optical information. The phase problem in optics appears with different faces depending on the optical phenomena investigated. The type of functional forms, sine or cosine, is crucial to determine the contrast of images, which is governed by the behavior of low‐frequency components in images. The charge‐inducing surface potential in the phase plate can be obtained by the comparison of contrast transfer functions (CTFs) with and without the phase plate. The phase plate itself is not charged when it is made of conducting material, such as carbon. There are three sources for the charge contamination: organic materials, metal oxides, and inorganic materials.

Complex Observation in Electron Microscopy.I.Basic Scheme to Surpass the Scherzer Limit

A novel observation scheme, termed the complex observation, which is faithful to reconstruct optical images carried by either electromagnetic or material waves in their intrinsic complex form, is proposed. This observation is based completely on the coherent microscopy and comprised of a triple experiment consisted of twin experiments restoring two linear terms corresponding to the real and imaginary part of complex images and an additional experiment to cancel the square term which is in company with either linear term. A linear combination of three images obtained through the triple experiment is able to exhibit a complex quantity in the form to be numerically manipulated without rupture according to the formal theory of image formation. Manipulation of the phase and the intensity of primary waves penetrating through objects is the key in this observation. The basic scheme is applied to settle the long-standing issue in electron microscopy that the point resolution is impaired by the modulated contrast ...

Complex Observation in Electron Microscopy: V. Phase Retrieval for Strong Objects with Foucault Knife-edge Scanning

Conventional phase retrieval in transmission microscopy is applicable only to weak objects that perturb the incidence with a phase that is π/2 or smaller. We propose a novel phase retrieval technique applicable to strong objects. The innovation core is the scanning of a knife-edge, which is conventionally fixed to recover phase information. The synchronous operation between the scanning of the knife-edge and the image accumulation enables a novel spatial filter, which draws phase retardations by objects in the form of their first derivative. Combining the left- and right-scanning of the knife-edge can completely expel image components that are non-linear to the wavefront functions. Theoretical formulation of knife-edge scanning filters and corresponding numerical simulations specific to an electron microscope are proposed.

Visualizing virus assembly intermediates inside marine cyanobacteria by zernike phase contrast electron cryo-tomography

1. National Center for Macromolecular Imaging, Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. 2. Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, USA. 3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 4. Department of Biology, Northeastern University, Boston, MA, USA. 5. National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Japan † Present address: FEI, 5350 Dawson Creek Drive, Hillsboro, OR, USA