In this study the technique of energy-filtering transmission electron microscopy was applied to localize cyanophycin (CGP) in recombinant strains of Ralstonia eutropha. Since CGP is a polymer consisting of the amino acids aspartate and arginine, which functions as a temporary nitrogen reserve that is deposited as insoluble inclusions in the cytoplasm of the cell, its nitrogen content is significantly higher than that of the other cell matter. In this study, we recorded nitrogen distribution maps, which represent the location of CGP in ultrathin sections of resin-embedded cells of recombinant strains of R. eutropha expressing the cyanophycin synthetase of Anabaena sp. strain PCC 7120. Furthermore, the existence of nitrogen in CGP granules was additionally proven by recording electron energy-loss spectra. The samples of R. eutropha H16 (pBBR1MCS-2::cphA1(7120)) revealed a second type of granule, which does not show nitrogen in the corresponding maps and which can be identified as an inclusion containing poly(3-hydroxybutyric acid). The methods applied in this study are suitable to identify storage compounds with elevated nitrogen contents and to reveal their location in the bacterial cell. The methods are also very helpful to distinguish between inclusions of different chemical compositions that occur both at the same time in the cells but cannot or only hardly be distinguished by other methods.

No abstract

In this study we analyse diffractograms of elastically filtered images of thin amorphous foils of carbon, silicon and germanium using the weak object approximation. The use of this approximation leads to a contrast transfer function containing a phase η(u) depending on the spatial frequency u. Furthermore, the derivative of this phase is included in the envelope function of the contrast transfer function. The phase can be attributed to the breakdown of the first-order Born approximation leading to complex scattering amplitudes characterized by this phase η(u). We analyse contrast transfer characteristics to determine the phase of complex scattering amplitudes of carbon, silicon and germanium as a function of spatial frequency and to measure the two-particle structure factor of the corresponding amorphous specimens. The contrast transfer characteristics were calculated from diffractograms of focal series of elastically filtered images. The phases measured show a decay with increasing spatial frequency and additional oscillations. The results for the two-particle structure factor also decay with increasing spatial frequency and contain low local maxima. Both can be attributed to voids or inhomogeneities within the amorphous structure.