Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system.

We prove that the focused ion beam (FIB) machine can be used as a microscope and as an in situ cutting device for tissue and cells. For the first time we obtain high-resolution ion images, complemented by electron imaging of different animal tissues both from uncoated and coated samples. In our study, we select the digestive system of Porcellio scaber (isopoda, crustacea) as a test system for FIB microscopy and in situ sample preparation. After the milling operation, some of the ultrastructural elements of hepatopancreatic cells can clearly be recognized. Also, FIB operation reveals significant differences in structural integrity between the apical and basal parts of hepatopancreatic cells, which have not been observed before by classical microscopy techniques. FIB microscopy and in situ sample preparation have advantages over classical microscopy techniques because of: 1. in situ site-specific 2-D sectioning and imaging of subsurface microstructures; 2. no need to embed the sample prior to sectioning; and 3. a wide range of magnifications while imaging the same sample.