Effects of dehydration and drying steps on human chromosome interior revealed by focused ion beam/scanning electron microscopy (FIB/SEM)

Abstract

Chromosome higher order structure has long remained unclear since the discovery of chromosome. So far, chromosome structure has been studied using electron microscopy because of its superior resolution and magnification. Recently, researchers started using focused ion beam/scanning electron microscopy (FIB/SEM) to obtain chromosome interior by simultaneous dissection and direct observation of the sections. To minimize distortion of ultrastructure of samples caused by rapid water evaporation under vacuum condition, a critical point drying (CPD) method including pre-dehydration has been extensively used. However, shrinkage or other artifacts in biological samples have also been reported using this method. On the other hand, the ionic liquid (IL) method has been developed to observe biological samples without dehydration, drying and metal/carbon coating by covering samples with a nonvolatile salt. In this study, the inner structure of isolated human chromosomes prepared using CPD and the IL method was observed by FIB/SEM. As a result, it became clear that the cavities appeared in chromosome only when CPD was applied during the preparation steps, and other steps such as fixation and dehydration may have less effect on the appearance of cavities compared to CPD. In conclusion, CPD method should be carefully used when the target is a very small biological sample such as chromosome.