Preparation of Membranes for TEM/SEM Techniques

All particle suspensions were prepared freshly before the experiments. Preparation and measurement of each nanoscaled suspension followed the methods described before (see chapter 4.2). Cells grown on microporous membranes were exposed for 1 hr, 24 hrs or 48 hrs from the apical side to each particle type using three different particle concentrations (10 ng/cm², 100 ng/cm² and 1000ng/cm²). Afterwards, all membranes were further processed for TEM analysis.

Preparation of membranes for TEM techniques

After 1 hr, 24 hrs or 48 hrs, cell samples exposed to test particles on microporous membranes were fixed in 5 % glutaraldehyde solution (pH 7.2) at room temperature for at least 1 hr. The PET-membrane was cut out and postfixed in 1 % osmium-tetroxid in 0.1M sodium cacodylate buffer. Following dehydration of the cells in a graded series of ethanol the membranes were infiltrated with epoxy resin and em-bedded. An ultramicrotome (Ultracut E, Richard-Jung) was used for cutting ultrathin sections (70nm). They were positioned on copper grids and observed with a trans-mission electron microsope (Zeiss, Leo 910).

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