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

Abstract

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.

Microscopic Analysis of the Interaction of Gold Nanoparticles with Cells of the Innate Immune System

“…If AuNPs detected in transmission mode were not detected on the surface, we proceeded to search inside the cell by performing a cut using FIB milling. Before the cut, and in order to protect the region of the cell containing the AuNPs and avoid the curtain effect (formation of striations across the milling face when the sample has a surface with uneven topography), we deposited a protective metallic layer on top using Pt-GIS activated with the electron gun (the preparation of the cut is shown in Figure 2 F highlighting the deposition with artificial shadowing) 19 . The cut was made using the smallest aperture of our FIB at 1.5 pA current to avoid damage caused by the FIB to the cells.…”

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

“…After drying, the cells were mounted on aluminum stubs and coated with gold by magnetron sputtering (Bal-Tec SCD 050 sputter coater). The samples were analyzed with an FEI Strata DB 235 M scanning electron microscope or a Sirion 400 NC focused-ion-beam/scanning electron microscope (17).…”

Focus Ion Beam/Scanning Electron Microscopy Characterization of Osteoclastic Resorption of Calcium Phosphate Substrates

“…Drobne et al, as well as other authors, extensively described the potential of FIB milling to process biological samples and shed light on the optimal processing parameters in order to avoid shrinkage, melting effect, Ga + implantation or side-wall artefacts. 18,[27][28][29][30] These effects can be effectively reduced by a first Pt layer deposition which protects the sample surface against re-deposition of ablated atoms, provides mechanical stability and reduces curtain effects. In addition, damage can be minimized by working with low ion beam currents and low acceleration voltages, especially during the final steps of the crosssection polishing.…”