Abstract. In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.

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High-pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze-substitution) of biological tissue up to a thickness of about 200 microns. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine-needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.

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Reactive high power impulse magnetron sputtering (rHiPIMS) was used to deposit silicon nitride (SiNx) coatings for bio-medical applications. The SiNx growth and plasma characterization were conducted in an industrial coater, using Si targets and N2 as reactive gas. The effects of different N2-toAr flow ratios between 0 and 0.3, pulse frequencies, target power settings and substrate temperatures on the discharge and the N content of SiNx coatings were investigated. Plasma ion mass spectrometry shows high amounts of ionized isotopes during the initial part of the pulse for discharges with low N2-to-Ar flow ratios of < 0.16, while signals from ionized molecules rise with the N2-to-Ar flow ratio at the pulse end and during pulse-off times. Langmuir probe measurements show electron temperatures between 2 -3 eV for non-reactive discharges and 5.0 to 6.6 eV for discharges in transition mode. The SiNx coatings were characterized with respect to their composition, chemical bond structure, density and mechanical properties by X-ray photoelectron spectroscopy, X-ray reflectivity, X-ray diffraction, and nanoindentation, respectively. The SiNx deposition processes and coating properties are mainly influenced by the N2-to-Ar flow ratio and thus by the N content in the SiNx films and to a lower extent by the HiPIMS frequencies and power settings as well as substrate temperatures. Increasing N2-to-Ar flow ratios lead to decreasing growth rates, while the N contents, coating densities, residual stresses and the hardnesses increase. These experimental findings were corroborated by density functional theory calculations of precursor species present during rHiPIMS.