Search

The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelat complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.

Acting through a cAMP-cAMP-dependent protein kinase (cAPK) cascade, members of two neuropeptide families, the small cardioactive peptides and myomodulins, modulate contraction amplitude and relaxation rate in the accessory radula closer (ARC) muscle of the marine mollusc Aplysia californica. An approximately 750-kDa phosphoprotein was identified in the ARC muscle as the major substrate for cAPK activated either by application of neuropeptides or by peptides released by motorneuron stimulation at physiological frequencies. Immunoblot and immunoelectron microscopy experiments revealed the widespread presence of this protein in Aplysia muscles and its colocalization with contractile filaments in the ARC muscle. Sequence analysis of proteolytic peptide fragments derived from the protein indicated that it is structurally related to the muscle protein twitchin. Finally, the level of neuropeptide-induced phosphorylation of the protein correlated well with peptidergic modulation of the relaxation rate of the muscle. We propose that twitchin in Aplysia, and perhaps in other species, may mediate the modulation of the relaxation rate of muscle contractions.

Despite their ubiquitous presence in the central and peripheral nervous systems, the behavioral functions of peptide co-transmitters remain to be elucidated. The marine molluscAplysia, whose simple nervous system facilitates the study of the neural basis of behavior, was used to investigate the role of peptidergic co-transmission in feeding behavior. Several novel modulatory neuropeptides were purified, and localized to identified cholinergic motorneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motorneourons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change.

We examined food and habitat use of sympatric smallmouth bass Micropterus dolomieui and rock bass Ambloplites rupestris in the Current and Jacks Fork rivers, Missouri. Use of food resources was similar between species and within a species. On a total caloric basis, the importance of prey ranked crayfish > fish > insects. The similarities of both prey types and the size selection of prey indicated that food resources were not a mechanism of ecological segregation between rock and smallmouth bass. Habitat use was evaluated by underwater observation. Location of both species was highly correlated with some aspect of cover, especially woody structure. Although cover use overlapped among size classes and species, rock bass were more often associated with rootwads and smallmouth bass with log complexes (aggregates of logs and branches). Cover use was independent of rock bass size, but smallmouth bass shorter than 350 mm long more often used vegetation and boulders and were more often found in open water than were smallmouth bass larger than 350 mm. Smallmouth bass used woody structure associated with higher current velocities more often than did rock bass. A positive relation between water depth and fish size was evident for each species, but water depth appeared unimportant in interspecific segregation. Received January 21, 1983 Accepted March 22, 1984

A procedure for growing hcp argon single crystals doped with oxygen is described. The hcp crystal structure is confirmed by birefingence and neutron scattering experiments. The birefringence at 77 °K of a hcp argon single crystal doped with 2% oxygen is determined to be ne-n0= + (2.11 ± 0.63) · 10-6. From this result one can calculate the Lorentz field factors, obtaining fo= (333 328.6 ± 1.4) · 10-6and fe= (333 342.7 ± 2.8) · 10-8. Using Ewald's theory of structural birefringence to determine σ = c/a from the measured birefringence yields σ = (163 451 ± 3) · 10-5. This value definitely excludes the ideal hcp structure (σ = 1.63299), in contrast with the results of X-ray measurements.

Road or railway tunnels are taken into account in noise prediction programs with the traffic flow ending at the tunnel mouth. The sound radiated from inside the tunnel through the tunnel mouth into the environment is calculated using an additional point or area source at the position of the tunnel opening. Some investigations have been published where the source strength of the radiating tunnel mouth has been calculated from analytically derived propagation relations inside the tunnel. In the meantime sound propagation inside rooms can be modelled more precisely even for complex environments, where the basic algorithms have been selected and improved based on measurements in more than 150 rooms and halls. These techniques have been standardized and in the meantime after about 8 years of experience with the application of these strategies they form an experimentally well established and computer based method that can be used to solve many of these special problems. The procedure is used as a part of an integral approach to calculate the noise radiated from tunnel openings. It can be used for different and even complex configurations and may help to integrate this very special noise source in environmental modelling for noise calculation. [ABSTRACT FROM AUTHOR]

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).

The strong technological trend towards higher and higher integrated systems is resulting in considerably increasing demands on nanotechnique and -electronics. One of the most important sets of information is the characterization of the inner interfaces within such materials not only concerning the atomic structure but also concerning chemical composition and bonding and concerning local strain. New generation energy filtering TEMs using corrected in-column spectrometers and providing considerably increased power of resolution concerning imaging and analysis are thus essential in order to be able to understand, define, built and characterize such devices.Within the SESAMe (Sub-eV-sub-Angstrom Microscope) [1] project, LEO and CEOS realize such a microscope for the MPI of Metal Research, Stuttgart in 3 steps. For step 1 and 2 an in second order completely corrected and in third order minimized 90° OMEGA energy filter has been developed. This filter is operating since one year in a 200 kV LEO EFTEM with LaB6 emitter (Step 1) [4]. Results will be presented by W. Sigle during this meeting. This filter will also be incorporated in the Sub-Angstrom-TEM (SATEM) with Cs corrector for the Research Center Jülich.

Transmission electron microscopes have been built along with and guided by technological opportunities since the last five decades. Even though there are some “workhorse” type of microscopes, these instruments are still more or less built from the technological viewpoint and less from the viewpoint of ease of use in a wide range of applications. On the other hand, leading edge applications are the drivers for the development and the use of leading edge technology. The result then is a “race horse” which is of very limited benefit in “Real world”.During the last decade computers have been integrated to build microscope systems. in most cases, however, computers still have to deal with obsolete electron optical ray path designs and thus, have to be used more to overcome the problems of imperfect optics and bad design of ray paths than to provide optimized “Real world” capabilities.

Modern SSCs combine low noise with high sensitivity and provide digital images with high dynamic and excellent linearity. Since these images are immediately available in a PC, SSCs have become a non-dispensable tool for on-line image processing and software driven automated TEM tuning [1]. Many experiments performed nowadays in the area of high resolution, low dose, holographic reconstruction, and EFTEM [2] would not be possible without the digital input coming from SSCs.Despite their excellent characteristics SSCs provide a restricted number of individual image points in respect to film. This turned out to be the most prominent disadvantage of SSCs especially for applications where highly resolved large specimen areas need to be captured. Tab. 1 shows a typical example for a biological application where an image detail of 4 nm (plasma membrane) needs to be resolved. Depending on the film material used, this resolution is matched more or less by a TEM magnification of ∼8,000x.

Excellent linearity and high sensitivity have made SSCs the ideal image detector for almost every TEM application. Their ability to make high quality digital images available within fraction of seconds for further evaluation and processing in a PC, have made them a non-dispensable accessory for any modern TEM. However, despite their excellent characteristics, SSCs provide a restricted number of individual image points in respect to a negative, what is considered to be the main disadvantage of this detector. To compensate for this, CCDs with 2048x2048 pixel are available since some time. SSCs using these 2kx2k CCD arrays not only provide 4 times the pixel number but also offer a lot more options people have waiting for: e. g. highly resolved low-dose or ESI images with significantly improved signal to noise ratio, or higher resolved images for diffraction analysis and holographic reconstruction.

The electron-optical design and the technical realisation of correctors, monochromators and imaging spectrometers as well as methods of resolution improvement (Focus variation, Holography) have made significant progress in the past few years. For all such devices and methods the mechanical and electrical stability of the microscope are the main challenges.Such requirements are even more critical in case of Energy Filtering Transmission Electron Microscopes (EFTEMs), where the achievement of highly resolved analytical results requires long detection times (in the range of a few 10s of seconds). Resolution in such case could mean both spatial and energy resolution even at the same time. The stability performance of the combined system is more complex than the sum of the stabilities of the single components. Thus, a specified HT short- and long-term stability of 2 ppm is not sufficient to achieve an energy resolution of 0.2 eV at 100 kV acceleration voltage.

Imaging of sample regions with a thickness significantly larger than the extinction length and strong thickness variations introduces two major problems for transmission electron microscopy (TEM) : (i) inelastic scattering increases the energy width of the transmitted electrons and therefore the resolution decreases (ii) the contrast differences caused by thickness variations can be higher than the dynamic range of the detector system.Both problems can be solved by using energy filtering techniques. The advantage here is that for energy filtered imaging the resolution limit is not determined by the sample thickness but by the width of the energy selection aperture. Fig. 1 shows three envelope functions of the temporal coherence calculated for different values of the energy width. The functions were plotted for an acceleration voltage of 200 kV and a high voltage stability of 2 ppm.

Recording of images under low dose conditions is a rather critical operation including many intermediate steps. Moreover, there is not a single fully agreed protocol concerning low dose operation but several competing ones potentially including individual variations. In any case, however, there should be a very well defined sequence of steps which must not allow for any mistake. Therefore tailored individual automation is the only way to obtain reliable results with maximum throughput at the same time.Such automation requires the functional integration of a fully remote controllable digital TEM, a Slow-Scan camera with a high sensitivity, and an acquisition/remote control software providing macro capabilities dedicated to different logical cameras. These requirements are met by LEO (EF)TEMs, by the ProScan SSCCD 14 bit camera, and the analySIS software 3.0.Three different logical cameras have to be configured in the SIS image processing software. The first camera is defined for searching for suitable specimen areas. It operates with 5 MHz and 8 bit image depth providing 1k×1k field size and resolution. The exposure time should be as short as possible, e.g. 50 ms for each frame. Via a preprocessing macro image brightness and EM magnification are set to the appropriate values, the desired shutter is activated, and the viewing screen is lifted for the exposure. A second postprocessing macro blanks the beam and does additional settings when the camera is stopped.

Since it's introduction into TEM in 1986 [1] SSCs have become an effective and easy-to-use solution for acquiring high quality digital images electronically. Their main advantages are excellent linearity, very high dynamic, high sensitivity, and low noise. These advantages have made SSCs a nondispensable tool for quantitative image analysis. Moreover, since SSCs make high quality TEM images available in a computer within fraction of seconds, on-line image processing and software driven automated TEM tuning has become possible [2]. Many of the experiments which are performed nowadays in the area of high resolution, low dose, holographic reconstruction, and EFTEM [3] would not be possible without the digital input coming from SSCs.Like recording images with a film sheet camera, digital recording of images with SSC requires control of the TEM beam blanker (shutter) by the SSC synchronised with the image acquisition process.This blanker (shutter) control is a critical link in the chain to the digital image. Many older TEMs do not have direct access to beam blanking coils, or contrary to modern TEMs their coils are not designed to support hysteresis-free fast beam blanking, what is essential for acquisition of high quality digital images by a SSC.

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 an EFTEM the full range of signals generated by interaction of the primary electron beam with the specimen can be detected. Thus operating such a system and generating combined digital information usually is a rather complex issue. The demands of the users on the other hand are to achieve results fast, easily and reproducibly. Moreover it should be possible to tailor the integral system according to dedicated needs. In general it should be no problem to use modern digital equipment and just let an integral computer control everything. However, in order to make such digital settings really useful, there should be no D/A conversion in between the data paths of the microscope because any analogue system tends to drift and the changes of lens parameters between different modes of operation should be minimised to overcome hysteresis. Fully digitised in-column EFTEMs like the LEO EFTEMs with OMEGA filter and Koehler illumination including also multiple parallel and serial remote capabilities provide optimum preconditions to fulfil the demands.

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 major demand for imaging light element specimens in the TEM is to achieve the appropriate contrast. This is particularly true in biomedical and also in polymer fields of application. The main reason for contrast being challenging is the correlation between the atomic number of the constituting elements and the scattering angle and thus the very low scattering absorption contrast achievable.Different conventional methods or combinations of them can be used to overcome the problem. 1) Contrast is created chemically by staining with heavy elements. Disadvantages besides the high level of preparational efforts are the artificial unnatural view in general and the high potential of artifacts in particular. 2) Contrast is created physically using small objective apertures and/or long focal length objective lenses. Disadvantages are clearly increased alignment and cleaning efforts for small apertures and the complete loss of high frequency/high resolution information. Moreover, some elastic scattering is still required. 3) Contrast is created physically converting phase contrast into absorption contrast using the phase contrast transfer function of the microscope and high defocus values.

The reaction of acridinone (I) and 1‐bromo‐3‐methylbut‐2‐ene (II) (molar ratio = 1:1) gives the desired glycocitrine‐II (III) and the derivatives (IV) and (V), whereas with an excess of (II) the pyranoacridinones (VI) and (VII) are formed.

The chloronitrobenzoic acid (I) is coupled with the dimethoxyaniline (II) to give the diarylamine (III) which is methylated to form the tertiary amine (V).

Eleven patients on chronic haemodialysis treatment thrice weekly received 1 μg 1,25(OH)2D3 i.v. after each dialysis for 3 weeks. Phosphate binders were mainly CaCO3, supplemented in a few patients by moderate amounts of A1(OH)3. Ionised calcium was measured by ion-selective electrode, normal values being 1.28–1.42mmol/l. PTH was estimated by an N-terminalsensitive assay; normal values are <0.25μg/ml. Results before and after 1,25(OH)2D3 were: ionised calcium before haemodialysis, 1.19±0.12and 1.17±0.14; ionised calcium after haemodialysis, 1.33±0.07 and 1.30±0.09; PTH before haemodialysis, 1.39±0.71 and 1.38±0.69; PTH after haemodialysis, 0.64±0.22 and 0.60 ±0.17; Phosphate before haemodialysis, 1.85±0.48 and 2.18±0.43 (P<0.05). No change of PTH concentration and ionised calcium before and after haemodialysis treatment could be documented after i.v. 1,25(OH)2D3 treatment. Mild and severe hyperparathyroidism were indistinguishable. Increased serum calcium concentrations therefore appear to be required for the suppression of PTH secretion by i.v. 1,25(OH)2D3 therapy.