Surface damage induced by FIB milling and imaging of biological samples is controllable.

Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.