Image-EELS for in situ estimation of the phosphorus content of RNP granules

Electron energy-loss spectroscopic imaging is performed on ultrathin sections, in order to approach the in-situ distribution of individual elements in the cellular constituents. In a previous ultrastructural study of salivary gland cells of Chironomus thummi and Ch. tentans by means of electron spectroscopic imaging and cytochemistry, we have described a new type of phosphorus-rich small granular ribonucleoprotein (RNP) component in the nucleoplasm of these cells; an attempt is made in the present work to estimate the phosphorus content of this component by the above mentioned method, using ribosomes as internal standard.   Results show that these RNP granules contain, on average 3600 phosphorus atoms per granule. Another nuclear constituent also studied, the Balbiani ring granule, contains, on average, 43 000 phosphorus atoms per granule.     

Videodensitometric analysis of electron spectroscopic micrographs — a tool for detection of biologically relevant elements with high resolution

Electron energy-loss spectroscopic imaging (ESI) yields high-resolution, element-sensitive images. However, ESI suffers from difficulties in distinguishing element-specific and background contributions. New methods have therefore been introduced which use grey-level measurements in micrographic images for a more accurate detection of element distributions. A videodensitometric method allowed the detection of low phosphorus levels in axoplasmic neurofilaments of squid giant axons. Here we further verify these results by investigating the relationship of videodensitometry and electron energy-loss spectroscopy (EELS), particularly considering the peculiarities of these methods in terms of automatic background correction and representation of the results. Six biological specimens and two nonbiological specimens were examined both by EELS and by videodensitometry. In all cases comparable results were obtained. The overlapping P_L2,3 and S_L2,3 ionization edges could clearly be recognized individually by both methods, and controls showed that mass density variations within the specimens did not impair elemental analysis. Additional evidence supporting the detection of phosphorylation sites in squid neurofilaments was obtained in both EELS and videodensitometric measurements of neurofilament-enriched pellets and of aggregated axoplasmic particles. Thus, videodensitometry appears to be a useful tool for an improved exploration of the full imaging capabilities of energy filtering electron microscopy.     

Ultrastructural aluminium detection in amphibian tissues by electron spectroscopic imaging and electron energy-loss spectroscopy

Aluminium causes a variety of toxic effects in living organisms but very little is known about its uptake, pathways and locations of deposition. We have applied electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) to locate aluminium at the ultrastructural level in amphibian larvae from acidic ponds. It is found diffusely bound or precipitated in cell organelles. The spatial resolution of aluminium detection is high. The elemental composition of small areas can be demonstrated by EELS. Three different fixation procedures give similar results. The two- and three-window methods at the K- and L-edges are compared.     

Experimental ionization cross-sections of phosphorus and calcium by electron spectroscopic imaging

The absolute partial electron scattering cross-section for the phosphorus L_2,3-shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross-section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂. At 80kcV, the partial cross-section of phosphorus was 2.26 times 10^?20 and 2.68 × 10^?20cm²/atom for poliovirus and hydroxyapatite, respectively, at 150eV loss for a 15-cV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross-section was 0.49 × 10^?20 cm²/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross-sections calculated either by hydrogenic or Hartree—Slater approaches.     

Elemental composition of pyroantimonate precipitates analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) in vitellogenic ovarian follicles of Drosophila

Ca²⁺ was precipitated with potassium antimonate in vitellogenic follicles of the fruit fly Drosophila melanogaster and the distribution of the precipitates formed was studied by electron microscopy. The microvilli of the oolemma in mid- and late vitellogenic follicles were lined with precipitates. The chemical composition of the precipitates was analysed by electron spectroscopic imaging (ESI). The images produced by inelastically scattered electrons at specific ionization edges were compared, and the nonspecific background signals were subtracted by an image processing system. The presence of Ca²⁺, antimony and oxygen in the precipitates formed could be demonstrated. The elemental composition of the precipitates and of yolk spheres was also analysed by electron energy-loss spectroscopy (EELS). With respect to the precipitates, signals at the calcium L_2,3-edge, the oxygen K-edge and the antimony M_4,5-edge were recorded without deconvolution and background subtraction. The yolk spheres, which were free of precipitates, gave the characteristic signal of the nitrogen K-edge. The applied techniques combine good ultrastructural resolution with the possibility of analysing the elemental composition of histochemical reaction products and cellular structures.     

Morphological studies of Cu(I)-complexed bipyridine-containing block copolymers by combined transmission electron microscopy techniques

The design of molecular species which can form well-defined macromolecular architectures by spontaneous, recognition-directed self-assembly of their components is a major goal in supramolecular chemistry. We synthesized block copolymers with polyether and bipyridine or oligo-(bipyridine) containing segments where the latter could form double-helical structures by metal complexation. Upon complexation with Cu(I) these systems showed a dramatic change in the thermal and mechanical properties from a waxy system to a thermoplastic elastomeric behaviour. This effect could be explained by the formation of a multiphase polymer system consisting of domains of bipyridine or oligo(bipyridine) Cu(I) complexes in a matrix of amorphous soft segments.
In the solution-cast films both nitrogen and copper could be detected with electron energy-loss spectroscopy. Element spectroscopic imaging pictures of the net nitrogen and copper element distribution were identical and revealed a microphase-separated system with identical morphological structures. High-resolution bright-field images revealed fine structures (interference lines) which were in accordance with stacks of [Cu(I)(bpy)₂] complexes. Electron diffraction patterns showed that the supramolecular order could be perfected through annealing.     

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

Visualization of water drying in porous materials by X‐ray phase contrast imaging

We present in this study results from X‐ray tomographic microscopy with synchrotron radiation performed both in attenuation and phase contrast modes on a limestone sample during two stages of water drying. No contrast agent was used in order to increase the X‐ray attenuation by water. We show that only by using the phase contrast mode it is possible to achieve enough water content change resolution to investigate the drying process at the pore‐scale. We performed 3D image analysis of the time‐differential phase contrast tomogram. We show by the results of such analysis that it is possible to obtain a reliable characterization of the spatial redistribution of water in the resolved pore system in agreement with what expected from the theory of drying in porous media and from measurements performed with other approaches. We thus show the potential of X‐ray phase contrast imaging for pore‐scale investigations of reactive water transport processes which cannot be imaged by adding a contrast agent for exploiting the standard attenuation contrast imaging mode.     

A study of Si-L and O-K ELNES in plant material: SiO2, Ca- and Zn-silicate in Minuartia

The formation of vacuolar precipitates containing silicon and calcium, as well as the accumulation of metal-silicates in cell walls of Minuartia has been investigated by electron microscopy, energy-dispersive X-ray analysis and electron spectroscopic imaging. The bond state of the elements concerned is examined by analysing energy-loss near-edge structures (ELNES), particularly the Si-L_2,3 and the O-K ELNES. Experimental ELNES results are compared with local densities of unoccupied states calculated by molecular orbital methods and results of XANES calculations.     

Quantitative and qualitative characterization of aquatic iron oxyhydroxide particles by EF-TEM

Electron energy-loss spectroscopy (EELS) and elemental imaging under the energy-filtered transmission electron microscope are powerful tools for the characterization of iron-rich particles present in natural waters. Features present in EEL spectra (Fe-M_2,3 Fe-L_2,3 and O-K ionization edges) of goethite (α-FeOOH) have been studied with an energy filter operated at 80 keV to determine optimal quantification and elemental imaging of Fe-rich natural aquatic particles in the 30–200 nm range of thickness. For quantitative aims, the Fe-M_2,3 ionization edge cannot be used easily, but the Fe-L_2,3 edge provides more accurate results owing to a better background extrapolation. The partial cross-section of the Fe(III) M shell has been determined for iron oxide. The use of two-windows (jump-ratio) and three-windows (background stripping) imaging methods is discussed in relation to the specimen thickness.