Biological specimen preparation for SEM by a method other than critical point drying

Abstract

Quality sample preparation for SEM is important to observe fine details without artifacts, and good preparation requires proper fixation, dehydration, drying and coating, An alternative 5 min passage in hexamethyldisilazane (HMDS) can replace critical point crying (CPD) and gives satisfactory results on many biological samples. CPD procedure may take up to 1 h per sample to ensure adequate drying, therefore a brief rinse in HMDS followed by air drying requires less time and equipment yet provides excellent results. Various biological samples were fixed in 3% glutaraldehyde; rinsed 3 times in Millonig's phosphate buffer for 10 min each; post-fixed in 1% osmium tetroxide for 1 h; rinsed as before; fixed again in 1% tannic acid (TA) for 30 min-1 h; rinsed well and partially dehydrated to 70% ethanol; placed in 1% uranyl-acetate (UA) in the dark, overnight: rinsed with 70% ethanol until UA cleared and then dehydrated through 100% ethanol.