Comparative analysis of specimen processing methods for spatial imaging of mulberry bud ultrastructure using the Scanning Electron Microscope

The transition of mulberry buds from a dormant state to active growth involves significant ultrastructural changes. Its critical observation advances our understanding of plant growth and regulations. Scanning Electron Microscopy (SEM) is uniquely capable of revealing such delicate ultrastructural details and developing an effective method for specimen preparation is therefore essential. Generally, recalcitrant biological samples need to be chemically fixed, consistently dried and uniformly coated with conductive material for its visualization in native state. The present study used mulberry dormant and active growing buds for testing different methods of specimen preparation involving combinations of hydrated samples, conductive coatings, standard chemical fixatives, and different drying techniques for best contrast meanwhile preserving the structures for SEM imaging. Our analysis confirmed that using glutaraldehyde (4%) as the primary fixative and post-fixing with osmium tetroxide (1%), followed by graded ethanol dehydration and hexamethyldisilazane (HMDS) mediated drying, is ideal for achieving remarkably high spatial resolution of bud images without damaging the native structural features. The results also highlight the importance of SEM imaging in detailing the internal features of the bud, including changes in the storage of metabolite, starch.