**4. Biological sample characterization**

FIB-SEM is the most advanced technique to be used for the analysis of biological samples; to do so, several protective measures need to be taken, since biological samples are always sensitive to heat, moisture, and pressure. For this reason, the biological samples must be fixated, stained, and embedded in resin. Since biological samples are large, that is, microscale, they need a lot of time to get processed. Image processing time is the biggest limiting factor during biological sample running through the FIB-SEM, since the machine has to scan each block one by one to reconstruct a full 3D image. Therefore, there should a balance between sample scanning time and good resolution and suitable contrast. To reduce the time factor and enhance the resolution and contrast, new methods like chemical fixation,

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**Figure 3.**

*Focused Ion Beam Tomography*

*DOI: http://dx.doi.org/10.5772/intechopen.88937*

**4.1 Chemical fixation treatment**

[54] and tannic acid [34] (**Table 1**, adopted from [33]).

with 2% uranyl acetate (aqueous) at room temperature.

**4.2 High-pressure freezing and freeze-substitution**

charging effect in FIB-SEM inspection of the samples.

high-pressure freezing, and freeze-substitution are being followed for sample preparation. **Figure 3** (Adopted from Wierzbicki et al. [47]) shows the interaction of Si nanowires with 3 T3 cell line. **Figure 3A** shows the nanowires taken up by the cell whereas **Figure 3B** shows the bending of nanowires under another cell [47].

Chemical fixation protocols use aldehyde fixation in the presence of uranyl acetate and osmium tetroxide by including thiocarbohydrazide osmium tetroxide

Currently, chemical fixation is the most widely used fixation technique among the researchers for FIB-SEM analysis of biological samples and high-resolution images can be recorded at a very small distance. At present, the large majority of FIB-SEM investigations are based on chemical fixation. For instance, animal liver tissue cell was examined under FIB-SEM and was fixated chemically with a chemical mixture of 2% OsO4 modified with 1.5% K3Fe3 + (CN)6 and 20% glutaraldehyde for 120 min at room temperature in phosphate buffer. Extra staining was done

Cryoimmobilization of samples for cryomicroscopy offers unique opportunities to inspect the sub-cellular structure in the absence of chemical fixation and metallic stains. Even though biological samples can be investigated under FIB-SEM for very clear images at room temperature [55], freeze substitution (FS) not only adds to the high conductivity of samples and high contrasting of images but also helps in preserving ultra-small structures when embedded with resin—because, during the FS processing, various desired chemicals and metallic agents can be put into the organic solvent to decrease the signal-to-noise ratio, resulting in the decline in the

Up until now, very few high-pressure freezing (HPF) and FS studies have been done for FIB-SEM sample preparation (**Table 2**, adopted from [33]). In one study [56], 24 different preparation protocols embracing HPF and FS techniques were used and no substantial difference was found in the contrast and structure of the cells. Another study did a comparative survey between the chemically and FS/HPFfixated mouse liver cell samples, whereas, for TEM, a mixture of glutaraldehyde and

*(Adopted from Wierzbicki et al. [47]) shows the interaction of Si nanowires with 3 T3 cell line. Figure 3A shows the nanowires taken up by the cell whereas Figure 3B shows the bending of nanowires under another cell.*

### *Focused Ion Beam Tomography DOI: http://dx.doi.org/10.5772/intechopen.88937*

*Ion Beam Techniques and Applications*

**Figure 2.**

*3.2.2 FIB/SEM tomography of porous Ni*

*XY-axis while sub image B shows its corrected version.*

internal caliber ranged from 500 to 2000 nm.

*3.2.3 FIB/EDS tomography of squeeze cast AlSi12*

each slice with the EDX (EDS) detector. Results can be seen in [12].

**4. Biological sample characterization**

milling direction. The Si in sample showed fibers and branches ~3–5 μm long with a diameter of 200–500 nm. These fibers are intermingled and represented by dif-

*Image of a cell (Adopted from Wierzbicki et al. [47]). Sub image A shows the shadow of trench onto cell in* 

calculated volume fraction of Si in choral structure in reconstructed region was ~5%.

SEM high-voxel resolution helped in architecting the 3D structure of porous nickel, refer to paper [12] for assistance. 3D imaging revealed that around 2/3% volume of selected area was porous with totally interconnected structure. The canal

FIB uses ion beam as working source, which is a result of interaction with sample

FIB-SEM is the most advanced technique to be used for the analysis of biological

samples; to do so, several protective measures need to be taken, since biological samples are always sensitive to heat, moisture, and pressure. For this reason, the biological samples must be fixated, stained, and embedded in resin. Since biological samples are large, that is, microscale, they need a lot of time to get processed. Image processing time is the biggest limiting factor during biological sample running through the FIB-SEM, since the machine has to scan each block one by one to reconstruct a full 3D image. Therefore, there should a balance between sample scanning time and good resolution and suitable contrast. To reduce the time factor and enhance the resolution and contrast, new methods like chemical fixation,

producing the secondary electrons, but when equipped with energy dispersive spectrometer, this can help in determining the elemental composition of each milled slice [29, 31]. The use of EDS with FIB resulted in finding the elemental composition of AlSi12 sample with each slice's elemental data. When dealing with complex chemical structures, this new hyphenation technique proved to be worthy to produce data with each slicing and give exact chemistry of the compound of interest. As far as sample is concerned, the EDS used 8-keV acceleration to analyze

. The finally

ference of colors. The interconnection junction sizes were 50–100 nm2

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high-pressure freezing, and freeze-substitution are being followed for sample preparation. **Figure 3** (Adopted from Wierzbicki et al. [47]) shows the interaction of Si nanowires with 3 T3 cell line. **Figure 3A** shows the nanowires taken up by the cell whereas **Figure 3B** shows the bending of nanowires under another cell [47].
