Focused ion beam (FIB) technology was first described in 1974 by Seliger and Fleming [1], who could focus an ion beam to a few micrometers. Later they demonstrated a scanning ion system with gallium ions that could be focused to 100 nm (review in ref. 2). After these initial approaches, the group of Dr. Ishitani at Hitachi [2, 3] refined the instruments and used them for sample preparation for transmission electron microscopy (TEM). The focused ion beam system was predominantly used in defect analysis in the semiconductor industry. At the spot of the defect material, an electron transparent lamella was prepared with the ion beam system to be analyzed at high resolution in the TEM [3–5]. Already in his review in 1996, Ishitani mentioned the possible use of the focused ion technology in biology [3].

The first instrument was a single-beam, ion beam microscope and later it was combined with an electron column. The ion beam is placed at an angle of 38° or 36° to the vertical electron beam and coincides at a working distance of about 4 mm. These instruments are called focused ion beam scanning electron microscopes, FIB-SEM (see Note 1).

Since the FIB-SEM technology entered the realm of biological applications about 10 years ago, it gained its merits in preparing TEM samples [6] from material that is difficult to cut, either by the hardness or large differences in hardness [5, 7] or incompatibility with diamond knives, e.g., titan implants in bone [8]. More recently, FIB-SEM was used to prepare thick cryo-sections for cry-TEM tomography [9–13].

Further, the ion beam can be used to cross-section cells or organisms prepared for scanning electron microscopy to access internal microstructures [2, 14–17] or to study cell-substrate interactions [18].

The most promising technology at the moment, however, is the FIB-SEM tomography [19–27]: From a sample embedded in resin, a cutting plane from the surface of the resin block is opened at a preselected region of interest. A thin section, as thin as 3 nm, is milled off in the ion beam and then with the electron beam, the freshly generated plane is imaged. By repeating this process, a 3D data set of the object under investigation is generated. Volumes usually in the range of 20–40 μm in all three dimensions can be imaged with a voxel resolution down to 3 nm. Up to now, FIB-SEM tomography resides between TEM tomography, with a volume of 2–5 μm in X, Y and 500 nm in Z, at around 1 nm resolution and 3View (Gatan, Inc.) tomography, using a microtome built in an SEM [28–31] with much larger volumes at 10–5 nm pixel resolution and about 30–50 nm Z slice resolution.

In this chapter we will concentrate on FIB-SEM tomography and the corresponding preparation methodology.

Biological samples require an extensive preparation protocol to stabilize the sample for electron microscopy and preserve the delicate ultrastructure. Further care has to be taken that the sample is well contrasted as no on-section staining can be applied.

In most cases this involves the final embedding into a block of resin, which is trimmed later in a way that the structures of interest become located close to the surface of the block. Often there are only a few ROI and they need to be identified within the large resin block. Sometimes the ROI can be located by secondary electron imaging directly from the relief of the block surface [23, 24] or with high acceleration voltages from heavy metal stained structures beneath the resin surface. For a precise location of specific cellular events, using coverslips with a reference grid [32] or placing landmarks [33] and correlative imaging is needed.

Here we like to introduce an alternative method, the thin layer plastification (TLP) technique, that allows for easy localization of features in cell monolayers or biofilms [34]. It follows the same preparation steps as block embedding, but instead of placing the sample in an embedding mould and burry it completely in resin, the bulk of the resin is removed before polymerization, which leaves just a thin resin layer surrounding the sample. This results in a surface showing some topography. The topographic information allows the selection of regions of interest in the FIB-SEM—even without previous correlation of multimodal images.

Biofilms on hard substrates like glass, e.g., a coverslip, require the removal of the support after conventional block embedding for being accessible by FIB-SEM. Thin layer plastification, on the contrary, allows the biofilms to stay on their substrate. They can be accessed directly from their apical side; thus, the cell-to-substrate interface remains intact and it can be visualized in situ. Thin layer plastification is, therefore, an ideal method for biological interface studies (see Fig. 1).