Comparison of different preparation methods of biological samples for FIB milling and SEM investigation.

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.

Long-term Hg pollution induced Hg tolerance in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea).

The aim of our work was to assess the pollution-induced community tolerance (PICT) of isopod gut microbiota and pollution-induced isopod population tolerance (PIPT). Animals collected from a chronically Hg polluted and an unpolluted location were exposed for 14 days to 10microg Hg/g dry food under laboratory conditions. The lysosomal membrane stability, hepatopancreas epithelium thickness, feeding activity and animal bacterial gut microbiota composition were determined. The results confirm the hypothesis that the response to short-term Hg exposure differs for animals from the Hg polluted and the unpolluted field locations. The animals and their gut microbiota from the Hg polluted location were less affected by Hg in a short-term feeding experiment than those from the unpolluted environment. We discuss the pollution-induced population tolerance of isopods and their gut microbiota as a measure of effects of long-term environmental pollution. The ecological consequences of such phenomena are also discussed.

Electron and ion imaging of gland cells using the FIB/SEM system.

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga(+) ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga(+) ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.