Prerequisites of High Resolution Scanning Electron Microscopy

Cryotechniques must be employed throughout all preparation and observation steps in order to extract high resolution scanning electron microscopical information from biological material. Cryoimmobilization, followed by freeze-drying and metal-shadowing at low temperature, yields optimal structural information of T4 polyheads used as a test specimen. Freeze-substitution of frozen T4 polyheads and subsequent freeze-drying renders the substructures recognizable but less crisp than freeze-drying from aequous solutions. Critical point drying of ethanol dehydrated chemically fixed, or freeze-substituted test specimens results in complete loss of discrete polyhead structure. In-lens field-emission scanning electron microscopes and highly sensitive electron detectors are instrumental prerequisites in achieving transmission electron microscope-like resolution of structural details. Shape and size of fine structures, of the test specimen, are accurately imaged at high acceleration voltage ( \u3e 7 kV). Precise localization of antigenic sites via ultra-small (0.8 nm) colloidal gold marker systems by backscattered electrons also depends on the appropriate choice of acceleration voltage. Contamination of the specimen surface is a serious problem in high resolution scanning electron microscopy. It can be controlled in practice, by photographing selected areas of cooled specimens during the first scan of the electron beam

Progress in Scanning Electron Microscopy of Frozen-Hydrated Biological Specimens

Modem scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens. This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples. Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g. accelerating voltage, electron beam irradiation of the specimen). Uncoated frozen-hydrated yeast cells do not charge electrically at an accelerating voltage of 30 kV. Increasing charging effects are however observed with decreasing accelerating voltages. Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal. Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope. Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained. Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail). Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions

Drugs, herbicides, and numerical simulation

The Colombian government sprays coca fields with herbicides in an effort to reduce drug production. Spray drifts at the Ecuador-Colombia border became an international issue. We developed a mathematical model for the herbicide aerial spray drift, enabling simulations of the phenomenon

Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli

The genes oadGAB encoding the oxaloacetate decarboxylase γ, α and β-subunits from Klebsiella pneumoniae were expressed in Escherichia coli. Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed α-subunit or a combination of a and β-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a γα-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the γα-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the a-subunit was not completely complexed to the membrane-bound γ or βγ-subunit

Synthese und spektroskopische Charakterisierung von Molybän(IV)-Oxo-Komplexen mit tetradentaten Phosphin-Liganden

Die vorliegende Arbeit befasst sich mit der Darstellung und spektroskopischen Untersuchung von Molybdän(IV)-Oxo-Komplexen mit polydentaten Phosphin-Liganden und deren Eignung für die synthetische Stickstofffixierung. Im ersten Abschnitt wird die Reaktivität von verschiedenen Molybdän(IV)-Oxo-Komplexen des Typs [Mo(O)X2(PMe3)3] gegenüber Austauschreaktionen mit polydentaten Phosphin-Liganden (1,2-Bis(phenylphosphino)ethan (mppe) und 1,1,4,8,11,11-Hexaphenyl-1,4,8,11-tetraphosphaundecan (prP4)) untersucht. Die Umsetzung des Liganden prP4 mit dem Komplex [Mo(O)I2(PMe3)3] lieferte selektiv den Komplex trans-[Mo(O)I(meso-prP4)]BPh4, der spektroskopisch und kristallographisch untersucht wurde. Der zweite Teil beschreibt die Darstellung der ersten Molybdän(0)-und Wolfram(0)-bis(distickstoff)komplexe mit einem P4-Liganden. Durch eine elektrochemische Reduktion des Komplexes trans-[Mo(O)I(meso-prP4)]BPh4 konnte selektiv der trans-Bis(distickstoff)komplex trans-[Mo(N2)2(meso-prP4)] dargestellt werden. Die direkte Umsetzung des prP4-Liganden mit MoCl5 bzw. WCl6 und Magnesiumspänen als Reduktionsmittel lieferte dagegen die trans- und cis-Bis(distickstoff)komplexe

Nanostructural organization of naturally occurring composites - part II: silica-chitin-based biocomposites

Investigations of the micro- and nanostructures and chemical composition of the sponge skeletons as examples for natural structural biocomposites are of fundamental scientific relevance. Recently, we show that some demosponges (Verongula gigantea, Aplysina sp.) and glass sponges (Farrea occa, Euplectella aspergillum) possess chitin as a component of their skeletons. The main practical approach we used for chitin isolation was based on alkali treatment of corresponding external layers of spicules sponge material with the aim of obtaining alkali-resistant compounds for detailed analysis. Here, we present a detailed study of the structural and physicochemical properties of spicules of the glass sponge Rossella fibulata. The structural similarity of chitin derived from this sponge to invertebrate alpha chitin has been confirmed by us unambiguously using physicochemical and biochemical methods. This is the first report of a silica-chitin composite biomaterial found in Rossella species. Finally, the present work includes a discussion related to strategies for the practical application of silica-chitin-based composites as biomaterials