**4.1 Chemical fixation treatment**

Chemical fixation protocols use aldehyde fixation in the presence of uranyl acetate and osmium tetroxide by including thiocarbohydrazide osmium tetroxide [54] and tannic acid [34] (**Table 1**, adopted from [33]).

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