DNA mapping in the capsid of giant bacteriophage phiEL (Caudovirales: Myoviridae: Elvirus) by analytical electron microscopy

Introduction. Giant phiKZ-like bacteriophages have a unique protein formation inside the capsid, an inner body (IB) with supercoiled DNA molecule wrapped around it. Standard cryo-electron microscopy (cryo-EM) approaches do not allow to distinguish this structure from the surrounding nucleic acid of the phage. We previously developed an analytical approach to visualize protein-DNA complexes on Escherichia coli bacterial cell slices using the chemical element phosphorus as a marker. In the study presented, we adapted this technique for much smaller objects, namely the capsids of phiKZ-like bacteriophages.Material and methods. Following electron microscopy techniques were used in the study: analytical (AEM) (electron energy loss spectroscopy, EELS), and cryo-EM (images of samples subjected to low and high dose of electron irradiation were compared).Results. We studied DNA packaging inside the capsids of giant bacteriophages phiEL from the Myoviridae family that infect Pseudomonas aeruginosa. Phosphorus distribution maps were obtained, showing an asymmetrical arrangement of DNA inside the capsid.Discussion. We developed and applied an IB imaging technique using a high angle dark-field detector (HAADF) and the STEM-EELS analytical approach. Phosphorus mapping by EELS and cryo-electron microscopy revealed a protein formation as IB within the phage phiEL capsid. The size of IB was estimated using theoretical calculations.Conclusion. The developed technique can be applied to study the distribution of phosphorus in other DNA- or RNA-containing viruses at relatively low concentrations of the element sought.

Comparative study on high-voltage nanosecond pulses and dielectric barrier discharge effects on surface morphology and physico-chemical properties of natural pyrrhotite

In this paper we used analytical electron microscopy, potentiometric titration (electrode potential), sorption and flotation measurements and other methods to study changes in the surface morphology, electrochemical, and physicochemical properties of the natural pyrrhotite exposed to nonthermal action of the repetitive nanosecond high-power electromagnetic pulses and low-temperature plasma of dielectric barrier discharge in air at atmospheric pressure. As a result of exposure to high-voltage nanosecond pulses, a sharp shift in the electrode potential of pyrrhotite to the region of negative values caused a decrease in the sorption of the anionic collector on the mineral, a decrease in the hydrophobicity of the surface and flotation of the mineral was due to an increase in the content of oxidized ferric iron on the mineral surface. Dielectric barrier discharge treatment caused the shift of the electrode potential to the region of negative values (–60 mV) in the range of pH 9.7-12, which causes the effect of a decrease in the sorption and flotation activity of pyrrhotite. The advantages of using the short-term (10-30 seconds) energy impacts for structural and chemical modification of the surface and physicochemical properties of sulfide minerals of iron are shown.

Textural and genetic relationships between glauconite and celadonite at the nanoscale: two different structural-compositional fields

Glauconite and celadonite coexist at the nanometre scale in Early Jurassic submarine volcanic rocks of the Betic Cordillera (southern Spain) as a result of microbial activity. Samples from the limit between the two micas, recognizable in scanning electron microscopy, have been extracted using the focussed ion beam technique and studied by high-resolution analytical electron microscopy. Both micas are present as randomly oriented differentiated small crystals in the boundary area. They define clearly distinct compositional fields with gaps affecting to Fe, Mg and K. At the lattice scale, celadonite shows a high degree of order, with homogeneous orientation of the visible lattice parameters being a difference from glauconite, formed by packets no more than 10-layers thick. Smectite layers were also detected alongside glauconite packets, in accordance with X-ray diffractograms which indicate that glauconite is a mica–smectite interstratification being more than 90 % mica layers. The compositional gap indicates that celadonite is not the endmember of the glauconitic series and the two micas represent two different structural tendencies of mica, with glauconite having more distorted octahedral sheets, indicated by systematically higher b parameters than celadonite.

Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy

Background: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cell (Danilova et al, 2020; Loiko et al, 2020). Here we applied this technique to study much smaller objects – the DNA packing inside bacteriophage heads. We studied phiEL, giant phiKZ-like bacteriophage of the Myoviridae family that infects Pseudomonas aeruginosa (Krylov et al, 2003). We have earlier demonstrated that this phage contains an ‘inner body’ inside its capsid, which is responsible for the specific DNA packing (Sokolova et al, 2014). Methods: The phage propagation was performed as described before (Sokolova et al, 2014). A 3 ul sample of purified bacteriophage phiEL was applied to the glow-discharged carbon-coated copper grid and stained with freshly prepared ammonium Molybdate 2% aquatic solution for 30 sec. Grids were loaded into Gatan cooling holder and the temperature of the specimen was kept at -180°C. EELS spectra and phosphorus elemental maps were obtained on JEOL2100 microscope, operating at 200 kV with the Gatan GIF Quantum ER spectrometer in STEM mode. Pixel size was set to 15-20 nm. STEM drift correction was applied after each 40-50 pixels. Each spectrum was obtained at a 6.0 mrad collection angle, 0.25 eV dispersion, and 132 eV energy shift. The spectra from different pixels were aligned to carbon K-edge. Results: Phosphorus mapping inside and outside the bacteriophage capsid was performed (Fig. 1). Outside the capsid, the phosphorus signal was practically absent, which corresponds to the presence of DNA only inside the capsid. The distribution of phosphorus inside the capsid was uneven: the rectangular area in the middle of the capsid contained a weak signal, while a more intense signal was detected on the periphery. This can be explained by the presence of an ‘inner body’ inside (Fig. 1C). Conclusion: Thus, our results justify the possibility of using the analytical EM technique to study the distribution of DNA by mapping Phosphorus in biological nano-objects at relatively low content of the element.

Principles of Materials Characterization and Metrology

Characterization enables a microscopic understanding of the fundamental properties of materials (Science) to predict their macroscopic behavior (Engineering). With this focus, the book presents a comprehensive discussion of the principles of materials characterization and metrology. Characterization techniques are introduced through elementary concepts of bonding, electronic structure of molecules and solids, and the arrangement of atoms in crystals. Then, the range of electrons, photons, ions, neutrons and scanning probes, used in characterization, including their generation and related beam-solid interactions that determine or limit their use, are presented. This is followed by ion-scattering methods, optics, optical diffraction, microscopy, and ellipsometry. Generalization of Fraunhofer diffraction to scattering by a three-dimensional arrangement of atoms in crystals, leads to X-ray, electron, and neutron diffraction methods, both from surfaces and the bulk. Discussion of transmission and analytical electron microscopy, including recent developments, is followed by chapters on scanning electron microscopy and scanning probe microscopies. It concludes with elaborate tables to provide a convenient and easily accessible way of summarizing the key points, features, and inter-relatedness of the different spectroscopy, diffraction, and imaging techniques presented throughout. The book uniquely combines a discussion of the physical principles and practical application of these characterization techniques to explain and illustrate the fundamental properties of a wide range of materials in a tool-based approach. Based on forty years of teaching and research, and including worked examples, test your knowledge questions, and exercises, the target readership of the book is wide, for it is expected to appeal to the teaching of undergraduate and graduate students, and to post-docs, in multiple disciplines of science, engineering, biology and art conservation, and to professionals in industry.