2.8. Scanning Electron Microscopy

Scanning electron microscopy (SEM) was determined using previously published methods [30] with minor modification; briefly, the SMM samples were cut into pieces of about 5 × 5 × 2 mm in size, and the treated SMM samples were fixed in 0.1% glutaraldehyde buffer solution for 1h and in 0.2% osmium tetroxide fixation solution for 10min. After, samples were washed with phosphate buffer for 3 times (10 min/time), dehydrated with gradient ethanol solution for 10 min each (25%, 50%, 70%, 80%, 90%, and 100%), and finally dehydrated with acetone for 10 min. The dehydrated SMM sample was placed in a 100 mL Centrifuge tube, which was frozen at −80 °C for 12 h and freeze-dried for 24 h. The freeze dried SMM sample was fixed on a bronze stub and sputtered with gold. It was then placed in a JSM-7800F scanning electron microscope (JEOL Ltd., Tokyo, Japan) with a 5 kV acceleration voltage to be observed with the scanning electron microscope (SEM) at 200 times and 1000 times magnification.