Optimization of water-rich starch sample preparation methods for scanning electron microscopy

In general, biological samples must be dehydrated and dried before high vacuum scanning electron microscopy (SEM) analysis. However, during the critical point-drying or freeze-drying treatment process, samples with high water content will shrink or collapse, whereas for starch rich plant samples, tissues are prone to starch loss and cell wall deformation, resulting in poor quality scanning images. By comparing three pre-treatment methods of SEM, including CO₂ critical point drying, freeze drying of chemically fixed samples, and freeze drying of fresh samples, we optimize suitable methods as follows:(1) Sample are first chemically fixed and then freeze dried, followed by brittle fracture with liquid nitrogen, with the cross-sections finally spray-coated with gold. With this treatment, the SEM images are of high quality, with the cell structure maintained and starch grains and protein complexes clear. This method can be used for analysis of the intracellular distribution of starch grains and protein. (2) Fresh samples are freeze dried directly, and after brittle fracture with liquid nitrogen, the cross-sections are finally spray-coated with gold. With this treatment, the SEM images are of high quality, with complete cell walls and clear starch granules. This is an optimal method for observation of starch granule distribution.