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

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 charging effect in FIB-SEM inspection of the samples.

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

### **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.*


### **Table 1.**

*Variation in chemical fixation protocols described in the literature to perform FIB-SEM tomography of biological samples.*


### **Table 2.**

*Variation in freeze-substitution protocols described in the literature for FIB-SEM tomography on biological samples cryofixed by HPF.*

methyl alcohol or acetone having hydrous or anhydrous uranyl acetate and OsO4 was used.

A study [57] has suggested that the difference in contrast between SEM and TEM images may be because of the fixatives, additive metals, and staining agents.

### **5. Conclusion**

It is concluded that FIB-SEM, FIB-TEM, and FIB-EDS are the latest techniques currently being used for the characterization as well as the tomographic studies of materials and biological samples.
