Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed

CHANGES OF THE SERUM ENZYME LEVEL IN PATIENTS WITH LIVER CARCINOMA

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Morphologies of synaptic protein membrane fusion interfaces

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release

3.9 angstrom structure of the nucleosome core particle determined by phase-plate cryo-EM

The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 angstrom and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors

Design of an electron microscope phase plate using a focused continuous-wave laser

We propose a Zernike phase contrast electron microscope that uses an intense laser focus to convert a phase image into a visible image. We present the relativistic quantum theory of the phase shift caused by the laser-electron-interaction, study resonant cavities for enhancing the laser intensity, and discuss applications in biology, soft materials science, and atomic and molecular physics.Comment: 5 pages, 3 figure

Steps towards the hyperfine splitting measurement of the muonic hydrogen ground state: pulsed muon beam and detection system characterization

The high precision measurement of the hyperfine splitting of the muonic-hydrogen atom ground state with pulsed and intense muon beam requires careful technological choices both in the construction of a gas target and of the detectors. In June 2014, the pressurized gas target of the FAMU experiment was exposed to the low energy pulsed muon beam at the RIKEN RAL muon facility. The objectives of the test were the characterization of the target, the hodoscope and the X-ray detectors. The apparatus consisted of a beam hodoscope and X-rays detectors made with high purity Germanium and Lanthanum Bromide crystals. In this paper the experimental setup is described and the results of the detector characterization are presented.Comment: 22 pages, 14 figures, published and open access on JINS

Experimental determination of the energy dependence of the rate of the muon transfer reaction from muonic hydrogen to oxygen for collision energies up to 0.1 eV

We report the first experimental determination of the collision-energy dependence of the muon transfer rate from the ground state of muonic hydrogen to oxygen at near-thermal energies. A sharp increase by nearly an order of magnitude in the energy range 0 - 70 meV was found that is not observed in other gases. The results set a reliable reference for quantum-mechanical calculations of low-energy processes with exotic atoms, and provide firm ground for the measurement of the hyperfine splitting in muonic hydrogen and the determination of the Zemach radius of the proton by the FAMU collaboration.Comment: 30 pages, 10 figure

First measurement of the temperature dependence of muon transfer rate from muonic hydrogen atoms to oxygen

We report the first measurement of the temperature dependence of muon transfer rate from muonic hydrogen atoms to oxygen between 100 and 300 K. Data were obtained from the X-ray spectra of delayed events in a gaseous target, made of a H2/O2 mixture, exposed to a muon beam. This work sets constraints on theoretical models of muon transfer and is of fundamental importance for the measurement of the hyperfine splitting of muonic hydrogen ground state as proposed by the FAMU collaboration