Imaging of biological structures with the scanning transmission electron microscope

The scanning transmission electron microscope (STEM) is discussed in view of biological applications. Theoretical considerations are given, but the emphasis is directed to practical examples from a range of biological projects. The STEM is most efficiently used in elastic and inelastic dark-field modes providing information on the scattering power of the irradiated sample. Thus, the STEM is an ideal tool for quantitative measurements such as mass-mapping or element-mapping at high resolution. Limitations of such methods due to multiple scattering and quantum noise are briefly reviewed.     

A new hypothesis for the evolution of biological electron transport

A new hypothesis for the evolution of biological electron transport is presented. According to this hypothesis biological electron transport originated close to the potential of the hydrogen electrode and evolved in various advantageous directions including, when molecular oxygen became available on the Earth, that of the oxygen electrode. This implies stepwise evolution along and across the potential scale. The hypothesis is based mainly on existing information obtained from studies of primary and tertiary structural relationships of proteins. It is hoped to provide a framework for closer understanding of both evolution and mechanisms of cellular oxidation-reduction as well as energy coupling reactions.     

Method to increase efficiency of irradiation of biological objects by electron beams

The method has been proposed for active control of distribution of the dose caused by an electron beam passed through the medium. The method is based on the influence of magnetic field on charged particles and it allows concentrating of the absorbed dose in the prescribed area. To investigate this method the Monte-Carlo simulations were carried out for 20-70 MeV electrons in 0.5-3 T magnetic field using GEANT program. From the obtained results it follows that in the uniform magnetic field the maximum in distribution of the electron beam dose appears and its shape is similar to Bregg's peak for heavy charged particles. The location of the maximum may be changed by varying beam energy and magnetic fields configuration. For 60-70 MeV beam the maximum may be obtained at the depth of 10-15 cm that is convenient for radiotherapeutic usage.     

Towards native-state imaging in biological context in the electron microscope

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.     

Ruthenium red-mediated osmium binding for examining uncoated biological material under the scanning electron microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% OsO4 and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Bisoliton mechanism of electron transport in biological systems

A nonlinear mechanism describing the transport of paired electrons in protein molecules is suggested. It is shown that electron-phonon interaction leads to the pairing of two electrons with local chain deformation in a state called a bisoliton. The parameters of the bisoliton and the stability conditions with respect to its decay into two-soliton state are estimated.I dedicate this paper to the memory of my teacher Professor A.S. Davydov, who attracted me to nonlinear science and was my colleague.     

A high-resolution electron microscope study of synthetic and biological carbonated apatites

A series of synthetic carbonated apatites and human dental enamels characterized by chemical analysis, infrared spectroscopy, and X-ray diffraction was studied using high-resolution transmission electron microscopy. Steps on the surfaces of apatite crystals, often only a few unit cells high and occasionally one unit cell high, were observed by their Fresnel diffraction contrast. Highly substituted synthetic carbonated apatites appeared to have more irregular and finer-textured surface features than materials with less carbonate substitution. The surface features of enamel apatite crystal were also irregular, but surface steps were less frequently aligned in crystallographic directions. Complex strain fields due to radiation damage centers were observed in some crystals and the fine structure of dislocations and grain boundaries in synthetic apatites was resolved at high magnification. Experimental lattice-image contrasts, in favorable circumstances, could be matched to computer-simulated images and were found to contain detail at near atomic resolution, around 2.0-2.5 A.