A promising aspect of ESI is its application in the detection of elemental labels introduced into biomolecules for cell and molecular biological techniques. Even though colloidal gold labeling for electron microscopy (EM) is highly developed, availability of alternative labels, especially for double or triple labeling applications would be helpful because of difficulties with gold concerning i) detection (gold diameters ≤1nm), ii) discrimination due to gold particle size variations in one size class, and iii) different labeling efficiencies depending on gold granule size. An alternative labeling molecule should contain a high concentration of a specific chemical element which is not or in minor concentrations present in the system under surveillance, and has to have the potential to be discriminated from “biological” elements by ESI. With respect to ESI, one candidate for elemental labeling is boron. It meets the criteria described above and substantial experience in the synthesis of labeling compounds exists. From the chemical point of view, the preferred labeling structure is a so called dendrimer, a highly branched regular three-dimensional monodisperse macromolecule. Dendritic structures offer a large variety of functionalities to incorporate an element detectable by energy filtering transmission electron microscopy (EFTEM).

In this study we report on the stabilization of short direct repetitive DNA elements. We arranged a 20 bp SK-primer element in a direct repeat manner within the cloning vector pBluescript KS (+). This resulted in an array of 27 direct repeats consisting of 24 bp units. We show that this plasmid could only be propagated without deletion of repeats in dam mutant Escherichia coli hosts, whereas all efforts to use strains that were defective in the methylation-dependent restriction system and the recA- or the mismatch repair-dependent deletion system failed. The deletions always affected whole repeat units and not parts of them, leading to an unpredictable reduction of the unit number down to a range of between 12 and two during propagation. We conclude that a Dam methylation-dependent, but recA- and mismatch repair-independent, deletion mechanism caused the DNA rearrangements without an obvious involvement of the known methylated-DNA restriction systems.

To image the distribution of a specific element in a specimen with an energy filtering TEM, the element-unspecific background under the core-edge has to be subtracted. The most commonly used procedure is the three-window power-law method leading to considerable systematic errors for low-energy core-edges. Here a new method is described which can be considered as a generalized difference method. Characteristic examples for element detection in biological specimens using this method are shown. The background under the core-edge can be described by one or two pre-edge windows as a polynome of third order. This function can be deduced from specimen areas that are not known to contain the element or from a second specimen used as a standard. Control experiments showed that background subtraction for on-overlapping core-edges in the low-loss region (50-200 eV) needs two pre-edge images, whereas at higher-energy losses (> 300 eV) only one pre-edge image is necessary. With the method described, objective elemental mapping becomes possible even for edges at 50-100 eV. This was proven for the M2,3-edge of iron at 60 eV. The detection of phosphorous was possible with a signal-to-noise ratio five times higher than when using the three-window method. Preliminary data showed that it should be possible to detect calcium with only one image before the edge.

Our investigations are aimed to exploit the potential of Electron Spectroscopic Imaging (ESI) using an LEO EM 912 Omega to perform element detection without any background signal included in the net elemental signal. In a first series of experiments we designed an approach for phosphorus (P) detection by using an internal mass-thickness marker in the shape of a carbon ramp combined with the two-window difference method in order to eliminate mass thickness signals [1]. However, pure carbon seems not to be ideal because this approach does not take into account the more complex elemental composition of biological samples ( H, N, O, P and S) compared to pure carbon within the reference. Consequently, we had to find an easy-to-handle and reproducible technique to include a combined Compositional- and Massthickness Marker (C:M-Marker) in the specimen under investigation. Fig. 1A shows the result of such an experiment. Turnip Yellow Mosaic Viruses (TYMV; bright particles) were layered on top of a carbon foil followed by a wash with distilled water and air drying. To form an internal C-M-Marker a 1%-solution of Bovine Serum Albumin (BSA), dialysed against H2O, was sprayed on the opposite side of the same sample resulting in localized aggregations of BSA with different thicknesses (bright region “M, Fig. 1A). It serves the same purpose like the carbon ramp [1]: a pre-edge image (115 eV) and a second one (150 eV, core edge image) are taken to calculate the intensity (I) background function I150 (I115) using only values from the C-M-Marker region. Then the P-map image can be calculated by subtracting the extrapolated contribution of the protein part from the core-edge image. For the case presented in Fig. 1A this procedure resulted in Fig. 1B where the TYMV intensity signals can still be seen whereas the BSA ramp vanished.

In ultrathin sections for electron microscopy the evaluation of images generated with immunocytochemical or in situ hybridization techniques can become difficult if the colloidal gold label is very small and the underlying structure shows a strong heavy metal contrast caused by conventional uranium/lead staining. Normally, the problem is overcome by analyzing specimens at higher magnifications or by enlarging the gold grains in a secondary process called silver enhancement. We present a method to visualize the small gold particles with electron spectroscopic imaging (ESI). Three energy-filtered images acquired at energy losses of 0, 45 and 120 eV are analyzed by digital image treatment so as to allow the optical separation of the Au-markers from the image background. This protocol can be adapted for applications related to the automated counting of the label.

ESI (electron spectroscopic imaging)-detection methods are mainly based on the core edges above 100 eV (see, as an example, Ref. 1). However, it would be of major interest to use the high intensity values in the low-loss region below 100 eV. There are at least two elements of general interest for biological applications showing core edges in this region: Au (O2,3 ) and Fe (M 2,3 )• Since colloidal gold and ferritin are used to label targets in biological samples at high spatial resolution we started our study aiming at iron-detection (iron-maximum at 62 eV comparable to gold-maximum at 65 eV).Ferritin (Ref. 2) contains about 200 to 2000 iron atoms in a densely packed core representing an ideal prerequisite for ESI-detection. Furthermore, ferritin used to be a common marker in conventional transmission electron microscopy in the past. The contrast produced by ferritin in a conventional transmission electron microscope (CTEM) is not specific for iron but only for a dense region in the specimen.

The element signal obtained from electron-energy-filtered micrographs depends on the systematic error in calculating the background and on the noise in the background-corrected image. Both systematic error and statistical fluctuation of the background can be assessed experimentally with a specimen that combines the element-containing feature with a mass-thickness marker. The approach is described for the mapping of phosphorus in turnip yellow mosaic viruses prepared on a supporting carbon film of variable thickness. The thickness modulations are produced by the additional deposition of heat-evaporated carbon through a second grid used as a mask. The three-window power-law method and the two-window difference method are compared. With the three-window power-law method, the mass-thickness modulations of the marker are still visible in the map, indicating a systematic error for the calculated background. In addition, the intensity profile over the area of the thick carbon film is broader than in the map corrected by the two-window method, indicating a higher level of noise. With the two-window difference method, mass-thickness contrast was practically eliminated due to an improved protocol that uses the mass-thickness marker to calculate the scaling factor: instead of scaling the grey-level of a single background feature, the pre-edge image is scaled to the contrast of the marker area in the image acquired at the element-specific energy loss.